H e r b e t t e Symposium on S p e c i e s Concept i n Hymenomycetes 1976

THE GENUS PLEUROTUS AS AN AID FOR UNDERSTANDING THE CONCEPT OF SPECIES IN BASIDIOfYlYCETES

A . B r e s i n s k y , 0 . H i l b e r * ) a n d H . P . M o l i t o r i s * * )

i . I n t r o d u c t i o n The s p e c i e s as b a s i c u n i t of s y s t e m a t i c d i v e r s i t y has been d e f i n e d f o r t h e h i g h e r f u n g i by g e n e r a t i o n s o f m y c o l o g i s t s on t h e b a s i s of o b s e r v a t i o n s upon m a t e r i a l c o l l e c t e d i n t h e f i e l d . In many c a s e s , houuever, c u l t u r a l e x p e r i m e n t s have been o m i t t e d , due t o d i f f i c u l t i e s i n o b t a i n i n g f r u i t b o d i e s . C a r e f u l o b s e r v a t i o n s and d e s c r i p t i o n s a r e i m p o r t a n t e l e m e n t s t o r e c o g n i z e the e v o l u t i o n a r y p a t t e r n s , they a r e , hoiuever, not s u f f i c i e n t per se t o u n d e r s t a n d the b i o l o g y of s p e c i e s . O b s e r v a t i o n s and d e s c r i p t i o n s , t h e r e f o r e , s h o u l d be supplemented by e x p e r i m e n t s f o r d e l i m i t a t i o n and d e f i n a b i l i t y o f s p e c i e s . In a d d i ­t i o n , q u e s t i o n s about t h e e v o l u t i o n a r y p r o c e s s of s p e c i a t i o n must be s o l v e d e x p e r i m e n t a l l y .

B e f o r e d e a l i n g w i t h t h e s p e c i a l s u i t a b i l i t y o f the genus P l e u r o t u s f o r the u n d e r s t a n d i n g o f t h e s p e c i e s c oncept i n h i g h e r f u n g i , we f i r s t would l i k e t o d i s c u s s some r e l e v a n t q u e s t i o n s and exemplary i n ­v e s t i g a t i o n s .

C o n c e r n i n g c o n s t a n c y and s t a b i l i t y o f t h e o b s e r v e d t a x o n o m i c f e a t u r e s a g a i n s t e n v i r o n m e n t a l c o n d i t i o n s , Gaumann (1923) has made i m p o r t a n t c o n t r i b u t i o n s by a number of c l a s s i c e x p e r i m e n t s . They a r e based on t h e c o n s i d e r a t i o n t h a t p h e n e t i c d i f f e r e n c e s may be m o d i f i e d by sup e r ­p o s i t i o n of e n v i r o n m e n t a l i n f l u e n c e s which makes i t d i f f i c u l t t o r e ­c o g n i z e t he d i f f e r e n c e s due t o g e n e t i c f a c t o r s . In f a c t , Gaumann 1s e x p e r i m e n t s on P e r o n o s p o r a have shown t h a t extreme f l u c t u a t i o n s o f t e m p e r a t u r e and h u m i d i t y i n c o n n e c t i o n w i t h g r e a t d i f f e r e n c e s i n age r e s u l t i n d e v i a t i o n s o f maximal 11 m i c r o n s i n the d i a m e t e r o f the co-n i d i a . T h i s e q u a l s a f t e r a l l h a l f t h e d i a m e t e r o f a normal c o n i d i u m

*) C u l t i v a t i o n of c a r p o p h o r e s , c r o s s i n g s and t h e i r e v a l u a t i o n by 0. H i l b e r w i t h t e c h n i c a l a s s i s t a n c e o f Mrs. R. lYlaier.

**) Growth e x p e r i m e n t s and enzyme s p e c t r a by H.P. ( Y l o l i t o r i s w i t h co­o p e r a t i o n o f A. S c h a r t l .

of P e r o n o s p o r a . I n the f i e l d , however, c h a r a c t e r - s h i f t i n g environmen­t a l c o n d i t i o n s w i l l h a r d l y p l a y t o g e t h e r i n t h a t way t h a t extreme de­v i a t i o n s r e s u l t from t h e s e .

I t i s an o t h e r q u e s t i o n t o what e x t e n t d i f f e r e n t s u b s t r a t e s a r e t o be made r e s p o n s i b l e f o r d i f f e r e n c e s i n the f e a t u r e s o f two compared f u n ­g i . The r o l e o f s u b s t r a t e - i n d u c e d m o d i f i c a t i o n s and t h e e x t e n t o f d i f f i c u l t i e s which a r i s e by them f o r the d e l i m i t a t i o n o f s p e c i e s i s c o n t r o v e r s i a l . C o n c e r n i n g t h e powdery mildews ( E r y s i p h a l e s ) and downy mildews ( P e r o n o s p o r a ) i t was p o s s i b l e by c r o s s i n o c u l a t i o n and by ob­s e r v a t i o n on m u t u al h o s t s t o e x c l u d e a h y p o t h e t i c a l l y p o s s i b l e i n ­f l u e n c e by d i f f e r e n t h o s t s (Blumer 1967, G u s t a v s s o n 1959). C o n t r a r y t o t h a t , o t h e r a u t h o r s ( S c h w e i z e r , 1919) o b s e r v e d s u b s t r a t e i n d u c e d m o d i f i c a t i o n s o f c h a r a c t e r s used as t a x o n o m i c c r i t e r i a i n o t h e r p a r a ­s i t i c f u n g i ( e . g . B r e m i a ) .

A p r o f o u n d u n d e r s t a n d i n g o f t h e s p e c i e s as t a x o n o m i c u n i t i s impos­s i b l e w i t h o u t i n f o r m a t i o n about t h e e v o l u t i o n a r y p r o c e s s e s which r e ­s u l t i n s p e c i a t i o n . V a r i a t i o n and s e l e c t i o n a r e nowadays commonly a c ­c e p t e d f a c t o r s i n e v o l u t i o n . F u r t h e r m o r e , f o r t h e t a x o n o m i s t whose job i s t o l o o k f o r d i s c o n t i n u i t i e s t h e i s o l a t i o n o f s p e c i e s i s a n o t h e r v e r y i m p o r t a n t , a l m o s t predominant complex o f f a c t o r s . T h i s , because due t o t h e d i f f e r e n t i s o l a t i n g mechanisms d e v e l o p i n g d i v e r g e n c i e s a r e s t a b i l i z e d and t h e r e f o r e t h e d i s t i n c t i o n of s p e c i e s becomes p o s s i b l e .

In t h e f u n g i , i n h i b i t i o n or p r e v e n t i o n o f f r e e m a t i n g by g e n e t i c i s o ­l a t i o n i s the main i s o l a t i n g f a c t o r i n the d i v e r s i f i c a t i o n of spe­c i e s . E c o l o g i c a l i s o l a t i o n , e.g. by s p e c i a l i z a t i o n f o r a c e r t a i n sub­s t r a t e or h o s t , i s i n t h i s c o n t e x t merely an a d d i t i o n a l f a c t o r . Only seldom, l i k e i n the case o f y e a s t s , i t seems t o be an i m p o r t a n t one. F a c t o r s p r e v e n t i n g m a t i n g become m a n i f e s t i n f u n g i p a r t i a l l y a l r e a d y b e f o r e a m o r p h o l o g i c a l or e c o l o g i c a l d i f f e r e n t i a t i o n may be o b s e r v e d ( e . g . between s t r a i n s o f P o d o s p o r a ; h e t e r o g e n i c i n c o m p a t i b i l i t y , E s s e r , 1 9 6 7 ) . In t h i s c o n t e x t the f u n g i d i f f e r from o t h e r o r g a n i s m s . Kemp (1975) even c o n s i d e r s g e n e t i c i s o l a t i o n t o be t h e f i r s t s t e p i n t h e e v o l u t i o n o f new s p e c i e s o f f u n g i and p r o p o s e s t h e f o l l o w i n g s e ­quence f o r the s i n g l e s t e p s of t h i s p r o c e s s : M u t a t i o n o f the genome -c y t o p l a s m a t i c i n c o m p a t i b i l i t y - p r e v e n t i o n o f m a t i n g - m o r p h o l o g i c a l and e c o l o g i c a l d i f f e r e n t i a t i o n . I t a g r e e s w e l l w i t h t h e i m p o r t a n c e o f

g e n e t i c i s o l a t i o n i n f u n g i t h a t i n t e r s p e c i e s h y b r i d s - b e s i d e a few e x c e p t i o n s ( B l a s t o c l a d i a l e s : A l l o m y c e s ; S a c c h a r o m y c e t a l e s ; S p h a e r i a -l e s : N e u r o s p o r a ; U s t i l a q i n a l e s ) - have been newer o b t a i n e d . T h i s f a c t i s not o n l y i n f a v o u r o f t h e e f f i c i e n c y of g e n e t i c i s o l a t i o n , but a l ­so s u p p o r t s t h e s p e c i e s c oncept o f t h e t a x o n o m i s t not b e i n g t o o n a r ­row. How w e l l t h e m i n u t i o u s and f o r the e x t e r n o b s e r v e r o f t e n s u s p i c i o u s way of t h e taxonomic work c o r r e s p o n d s w i t h the phenomenon of g e n e t i c i s o l a t i o n s h a l l be shown by t h e s t u d i e s of Lamour (1965 and 1972) on a l p i n e s p e c i e s of C l i t o c y b e ( F i g . l ) .

V a r i a t i o n i n c l u d e s p r o c e s s and r e s u l t of any change w i t h i n t h e genome and l e a d s t o d i f f e r e n c e s w i t h i n a s e r i e s or a p o p u l a t i o n . V a r i a t i o n i n c l u d e s a l s o t h e p a t t e r n of c h a r a c t e r d i s t r i b u t i o n among the i n d i v i d u a l s o f a p o p u l a t i o n or a s p e c i e s . Changes may be caused by gene m u t a t i o n s . W i t h i n t h e c o n t e x t o f s p e c i a t i o n , however, t h e f r e q u e n c y o f m u t a t i o n s d u r i n g e x p e r i m e n t a l work w i t h f u n g i i s o f t e n o v e r e s t i m a t e d , s i n c e the p r o c e s s of n a t u r a l s e l e c t i o n i n i t s e l i m i ­n a t i n g and t h e r e f o r e s t a b i l i z i n g f u n c t i o n t e n d s t o be u n d e r e s t i m a t e d . However i t may be p o s s i b l e t h a t changes by gene m u t a t i o n (spontaneous

-7 -5 r a t e o f m u t a t i o n i n f u n g i 1 x 10 t o 1 x 10 a c c o r d i n g t o E s s e r anc Kuenen, 1965) become d e c i s i v e f o r e v o l u t i o n i n the l o n g r u n , s i n c e f u n g i show such a h i g h number of n u c l e i and. n u c l e i c d i v i s i o n s b e f o r e u n d e r g o i n g caryogamy. U n f o r t u n a t e l y , changes i n chromosome number, e.g. by p o l y p l o i d i z a t i o n , i n f u n g i can h a r d l y be o b s e r v e d or i n ­d i r e c t l y v i a d i f f e r e n c e s i n n u c l e u s volume. A change i n the genome may a l s o be a c c o m p l i s h e d by r e c o m b i n a t i o n d u r i n g t h e s e x u a l c y c l e ac­c o r d i n g t o t h e p o s s i b i l i t i e s g i v e n by t h e r e s p e c t i v e m a t i n g system. ( E s s e r , 1 9 7 4 ) . F i n a l l y , one has t o keep i n mind t h e changes by m i t o ­t i c r e c o m b i n a t i o n f o l l o w i n g p a r a s e x u a l p r o c e s s e s , e s p e c i a l l y s i n c e such p r o c e s s e s have been shown t o e x i s t a l s o in. b a s i d i o m y c e t e s ( C o p r i n u s : Prud'Homme, 1963; S w i e z y n s k i , 1 9 6 3 ) .

S e l e c t i o n may have as w e l l a s t a b i l i z i n g (under f a i r l y con­s t a n t e n v i r o n m e n t a l c o n d i t i o n s ) as a l s o a p r o m o t i n g f u n c t i o n i n the e v o l u t i o n o f new s p e c i e s . P r o m o t i n g f a c t o r f o r e v o l u t i o n means i n t h i s c o n t e x t t h a t d i f f e r e n c e s which o c c u r i n p o p u l a t i o n s a r e enhancec by s e l e c t i o n under v a r i a b l e and c h a n g i n g e n v i r o n m e n t a l c o n d i t i o n s . The e x t e n d e d d i c a r y o p h a s e o f h i g h e r b a s i d i o m y c e t e s r e s u l t s i n a spe­c i f i c mechanism o f s e l e c t i o n . A c c o r d i n g t o our p r e s e n t knowledge i n a d j c a r y o n , not o n l y t h e two n u c l e i w i t h i n one d i c a r y o t i c h y p h a l com-

partment but a l s o t h e p a i r s of n u c l e i i n d i f f e r e n t h y p h a l compart­ments may d i f f e r g e n e t i c a l l y . A d i c a r y o n t h e r e f o r e may r e p r e s e n t a g e n o t y p i c m o s a i c ( B u r n e t t and P a r t i n g t o n , 1957). As i n d i c a t e d above, d i f f e r e n t p a r t s of a mycelium may be g e n e t i c a l l y d i f f e r e n t and i f r e a c h i n g d i f f e r e n t s u b s t r a t e s they a r e exposed t o d i f f e r e n t e n v i r o n ­m e n t a l c o n d i t i o n s . F o r c e s of s e l e c t i o n a c t i n g upon one s i n g l e myce­l i u m t h e r e f o r e may be h e t e r o g e n e o u s and may l e a d t o d i f f e r e n t r e s u l t s by v i r t u e o f the above mentioned mosaic s t r u c t u r e of the mycelium. The d i p l o i d n u c l e i which a r e formed i n the hymenium a r e a l r e a d y o p t i ­mized by t h e s e l e c t i v e p r o c e s s e s b e f o r e f u s i o n o f the n u c l e i t a k e s p l a c e . Depending on d i f f e r e n t c o n d i t i o n s of s e l e c t i o n , g e n e t i c a l l y d i f f e r e n t f r u i t b o d i e s may a r i s e from one mycelium. The e x t e n s i o n of t h e d i c a r y o t i c phase i n t h e l i f e c y c l e of b a s i d i o m y c e t e s enhances the e f f i c i e n c y of s e l e c t i o n and r e d u c e s the l o s s o f i n d i v i d u a l m y c e l i a .

In the f o r m a t i o n , l i m i t a t i o n and s t a b i l i z a t i o n o f s p e c i e s a l l t h r e e f a c t o r s , v a r i a t i o n , s e l e c t i o n and i s o l a t i o n p a r t i c i p a t e , o f t e n even i n a n t a g o n i s t i c i n t e r a c t i o n . Knowledge of the i n t e r a c t i o n o f the v a ­r i o u s e v o l u t i o n a r y f a c t o r s s h o u l d form t h e n e c e s s a r y g u i d e l i n e f o r t h e t a x o n o m i s t s work which o f t e n s t r e s s e s more p r a c t i c a l a s p e c t s . Gaumann (1923) d u r i n g h i s work on t h e downy mildews has r e c o g n i z e d t h e i m p o r t a n c e o f e x p e r i m e n t a l work f o r t h e s o l u t i o n o f t a x o n o m i c p r o b l e m s . The r e s u l t s t h u s o b t a i n e d f o r one t a x o n o m i c group, however, do not n e c e s s a r i l y a p p l y t o o t h e r s . I t i s t h e r e f o r e n e c e s s a r y t o l o o k f o r each group o f f u n g i f o r t h e s u i t a b l e r e p r e s e n t a t i v e s t o work w i t h . I t w i l l be shown here t o what e x t e n t the genus P l e u r o t u s i s s p e c i a l l y s u i t e d t o r e a c h an u n d e r s t a n d i n g of the s p e c i e s w i t h i n t h e h i g h e r b a s i d i o m y c e t e s .

The genus P l e u r o t u s c o m p r i s e s m o s t l y wood-decaying s p e c i e s , more s e l ­dom s a p r o p h y t e s on o t h e r p l a n t m a t e r i a l . P l e u r o t u s s p e c i e s may be c u l t i v a t e d i n the l a b o r a t o r y and f r u i t b o d i e s may be o b t a i n e d under c e r t a i n c o n d i t i o n s w i t h i n 4 (3-6) weeks. fYlonocaryotic m y c e l i a f o r ma­t i n g e x p e r i m e n t s may be produced from s p o r e s . The genus P l e u r o t u s i n ­c l u d e s s e v e r a l complex groups whose members o c c u r i n Europe under v e r y d i f f e r e n t e c o l o g i c a l c o n d i t i o n s from L a p l a n d i n t h e n o r t h t o S i c i l y i n the s o u t h and which a l s o show e x t e n s i v e v a r i a t i o n o f cha­r a c t e r i s t i c s . S i n c e f r u i t b o d i e s may be o b t a i n e d i n t h e l a b o r a t o r y w i t h i n a r e l a t i v e l y s h o r t t i m e one i s a b l e to s t u d y t h e i n f l u e n c e o f e n v i r o n m e n t a l c o n d i t i o n s upon t h e e x p r e s s i o n o f t a x o n o m i c a l l y r e l e -

want c h a r a c t e r s . The p r e r e q u i s i t e s a r e g i v e n t o study the p r e s e n c e or even the development of m a t i n g b a r r i e r s i n comparison t o the v a r i ­ous d e g r e e s of m o r p h o l o g i c a l and e c o l o g i c a l d i v e r g e n c y and t o o b t a i n i n s i g h t i n t o t h e p r o c e s s l e a d i n g t o s p e c i a t i o n .

2. S u r v e y o f t h e P l e u r o t u s - o s t r e a -t u s - c o m p l e x i n E u r o p e

A p r o p e r t a x o n o m i c t r e a t m e n t of the complex of P̂ . o s t r e a t u s i s not y e t r e a c h e d . On t h e one s i d e a t t e m p t s t o d i v i d e the complex and t o e v a l u a t e the rank of t h e u n i t s are s t i l l c o n t r o v e r s i a l , on the o t h e r s i d e the t o t a l d i v e r s i t y has not y e t been r e c o r d e d c o m p l e t e l y . A g r e a t s t e p f o r w a r d i n t h e taxonomy of t h i s group was done by Romagnesi (1969) t h r o u g h c o n s i d e r a t i o n o f a number o f m i c r o s c o p i c a l c h a r a c t e r s which had been n e g l e c t e d h i t h e r t o . The p r o v i s i o n a l key g i ­ven a t t h e end o f t h i s paper p r e s e n t s a s u r v e y o f t h e s p e c i e s which can be d i s t i n g u i s h e d a t the moment.

U n l i k e the group of P_. e r y n q i i , the s p e c i e s complex around P_. o s t r e a ­t u s does not show a narrow s p e c i f i c t y f o r d i f f e r e n t s u b s t r a t e s .

3. R e a l i z a t i o n a n d m o d i f i c a t i o n o f t a x o n o m i c c r i t e r i a u n d e r l a b o r a ­t o r y c o n d i t i o n s

Our own i n v e s t i g a t i o n s about the r e a l i z a t i o n of c h a r a c t e r i s t i c s under v a r i o u s e n v i r o n m e n t a l f a c t o r s a r e o n l y at an i n i t i a l s t a g e . Utmost c a r e must be a p p l i e d f o r i n t e r p r e t a t i o n . The p r e l i m i n a r y r e s u l t s , r e ­p o r t e d h e r e , show merely the tendency of f u r t h e r s t u d i e s . E x p e r i m e n t s and o b s e r v a t i o n s a r e d i s c u s s e d s e p a r a t e l y f o r some f e a t u r e s which are used i n t h e key f o r t h e d i s t i n c t i o n of s p e c i e s .

C o l o r o f c a p : In our e x p e r i m e n t s d i f f e r e n t s u b s t r a t e s d i d not e f f e c t t h e c o l o r o f the cap. UJe used s t r a i n 2y, o r i g i n a t i n g from f r u i t b o d i e s grown an S a l i x . The s t r a i n was i d e n t i f i e d as P_. os­t r e a t u s and produced i n c u l t u r e a l s o f r u i t b o d i e s on stumps of P i c e a and B e t u l a . Under any c i r c u m s t a n c e s t h e c o l o r of the cap was grey brown and was r a t e d as 5F3 a c c o r d i n g t o (Ylethuen * ) . T h i s c o l o r was

*) Kornerup and UJanscher 1967.

i d e n t i c a l w i t h t h a t o f the f r u i t b o d i e s on S a l i x a t the o r i g i n a l s i t e . S i n c e t h i s f u n g u s , o r i g i n a l l y f r u i t i n g on S a l i x , produced f r u i t b o d i e s a l s o on P i c e a and B e t u l a i t becomes e v i d e n t t h a t i t i s doubt­f u l t o base P_. s a l i q n u s and P_. o s t r e a t u s i n t h e key o n l y on d i f f e r e n t s u b s t r a t e s .

I f f r u i t b o d i e s were produced i n the l a b o r a t o r y under a r t i f i c i a l l i g h t of r e l a t i v e l y low i n t e n s i t y or on wheat s t r a w or wheat g r a i n s , they showed p a l e r c o l o r s . UJhen f r u i t b o d i e s were grown i n open p e t r i -d i s h e s w i t h wheat s t r a w i n a humid chamber, they had a y e l l o w i s h grey c o l o r (lYlethuen 4B2), whereas the f r u i t b o d i e s e x t e n d i n g from the r i m of E r l e n m e y e r - f l a s k s f i l l e d w i t h wheat g r a i n s were b r o w n i s h grey (lYlethuen 5D4). T h i s a g r e e s w i t h o b s e r v a t i o n s o f E g e r , Eden and l i i i s s i g ( 1 9 7 6 ) , who i n a d d i t i o n d e t e r m i n e d the i n f l u e n c e o f t e m p e r a t u r e . They no t e d a d a r k e r c o l o r o f t h e f r u i t b o d i e s w i t h l o w e r t e m p e r a t u r e s and w i t h h i g h e r l i g h t i n t e n s i t i e s .

The c o l o r s used i n t h e key f o r d i s t i n c t i o n of s p e c i e s remain a l s o un­der l a b o r a t o r y c o n d i t i o n s f a i r l y c o n s t a n t i f f r u i t i n g o c c u r s under c o n s t a n t and s u f f i c i e n t l i g h t (minimum 500 L u x ) . S t r a i n s o f P. p u l -m o n a r i u s r e t a i n t h e n t h e i r l i g h t , o f t e n a l m o s t w h i t i s h c o l o r and P_. c o l u m b i n u s w i l l a l w a y s be d i s t i n g u i s h e d from P_. o s t r e a t u s by the co­l o r of i t s cap. I f , however, f r u i t b o d i e s o f the d i f f e r e n t s p e c i e s were produced under d i f f e r e n t c o n d i t i o n s t h e c o l o r may become s i m i ­l a r . Changing e n v i r o n m e n t a l c o n d i t i o n s t h e r e f o r e m o d i f y t h e c o l o r d i f f e r e n c e s between the s p e c i e s . As l o n g as the n a t u r a l c o n d i t i o n o f f r u i t i n g i s f a i r l y w e l l i n c o n c o r d a n c e w i t h the n a t u r a l s i t u a t i o n , t h e r e i s o n l y a s h i f t , not a l o s s o f t h e s p e c i f i c d i f f e r e n c e s i n co­l o r .

T h e s h a p e o f c a p may be d i f f e r e n t a c c o r d i n g t o t h e c u l t u r e method. By h i g h e r h u m i d i t y , as i s g i v e n w i t h i n an E r l e n m e y e r -f l a s k w i t h s u b s t r a t e , f r u i t b o d i e s a r e produced which a r e f u n n e l r s h a -ped and whose caps a r e more l o b a t e d , the m argin b e i n g t r a n s p a r e n t l y s t r i a t e and t u r n e d upward. The caps under t h e s e c o n d i t i o n s a r e not seldom hygrophanous. E x p e r i m e n t s where the f r u i t b o d i e s a r e produced l a t e r a l l y on E r l e n m e y e r - f l a s k s s i m u l a t e most c l o s e l y n a t u r a l c o n d i ­t i o n s . Here the shape of the f r u i t body, th e c e n t r a l d e p r e s s i o n o f t h e cap, t h e i n v o l u t i o n o f t h e m argin and o t h e r t a x o n o m i c c r i t e r i a a r e not changed ( e . g . shape of cap o f P. c o r n u c o p i a e ) .

L e n g t h o f s t i p e : L i g h t i n t e n s i t y p r o ved t o be impor­t a n t f o r the l e n g t h of s t i p e . Z a d r a z i l and S c h n e i d e r e i t (1972) showed t h a t f r u i t b o d i e s grown under low l i g h t i n t e n s i t i e s produce l o n g s t i ­pes and the f r u i t b o d i e s f i n a l l y assume t h e h a b i t u s o f P_. c o r n u c o -p i a e . J a b l o n s k y (1975) c o u l d c o r r e l a t e l e n g t h o f s t i p e w i t h l i g h t i n ­t e n s i t i e s . The f r u i t b o d i e s show under d e f i n e d l i g h t i n t e n s i t i e s a c h a r a c t e r i s t i c l e n g t h o f s t i p e f o r each s t r a i n , however, i f t h i s p r o ­p e r t y i s compared under v a r i a b l e l i g h t i n t e n s i t i e s , t h e o v e r l a p p i n g of the l e n g t h o f s t i p e s i s r a t h e r l a r g e . From t h e s e and our own ob­s e r v a t i o n s f o l l o u s , t h a t l e n g t h o f s t i p e s can be used as t a x o n o m i c c r i t e r i o n o n l y under d e f i n e d and l i m i t e d c o n d i t i o n s . A g e n o t y p i c d i f ­f e r e n t i a t i o n i s here - as i t i s o f t e n t h e case - o b s c u r e d by m o d i f i ­c a t i o n s .

S c l e r i f i e d h y p h a e : Romagnesi (1969) p a i d s p e c i a l a t t e n t i o n a l s o t o the p r e s e n c e of s c l e r i f i e d hyphae i n c h a r a c t e r i z i n g P_. p u l m o n a r i u s and p a r t i a l l y a l s o P_. c o r n u c o p i a e . A c c o r d i n g t o our own o b s e r v a t i o n s , however, t h i s c r i t e r i o n i s o n l y o f l i m i t e d v a l u e , s i n c e i t i s r a t h e r e a s i l y m o d i f i e d . Depending on c u l t u r e c o n d i t i o n s s c l e r i f i e d hyphae i n f r u i t b o d i e s may be p r e s e n t or may be t o t a l l y a b s e n t . T h i s phenomenon was a l r e a d y d e m o n s t r a t e d by i n v e s t i g a t i o n o f one s i n g l e s t r a i n o f P_. o s t r e a t u s (2 y ) , whose f r u i t b o d i e s i f grown on sawdust of Faqus produced many s c l e r i f i e d hyphae, whereas f r u i t b o d i e s from wheat s t r a w , stumps of B e t u l a and P i c e a as w e l l as the o r i g i n a l c o l l e c t i o n from S a l i x d i d not c o n t a i n t h i c k w a l l e d hyphae i n t h e c o n t e x t .

In s t r a i n 1 w o f P_. o s t r e a t u s we found s c l e r i f i e d hyphae i n f r u i t bo­d i e s from wheat s t r a w , but not i n t h o s e grown on wheat g r a i n s ( F i g . 2 ) . P o s s i b l y a l s o o t h e r f a c t o r s t h a n s u b s t r a t e may be i m p o r t a n t f o r the o c c u r r e n c e o f s c l e r i f i e d hyphae. S t a n k o w i c o v a (1973) e.g. s t a t e s t h a t e s p e c i a l l y o l d e r f r u i t b o d i e s o f P_. o s t r e a t u s c o n t a i n t h i c k w a l ­l e d hyphae ( F i g . 2 ) .

D i m e n s i o n o f s p o r e s : U n t i l now d i m e n s i o n o f spo­r e s has not been c o n s i d e r e d f o r d i s t i n g u i s h i n g s p e c i e s w i t h i n t h e P l e u r o t u s - o s t r e a t u s - c o m p l e x , a l t h o u g h d i f f e r e n t maxima of s pore l e n g t h i n d i c a t e t h a t s t a t i s t i c a l t r e a t m e n t of s pore measurements c o u l d r e s u l t i n r e l e v a n t d i f f e r e n c e s of s pore s i z e . To see whether s p o r e s i z e c o u l d be used f o r t a x o n o m i c p u r p o s e s i n P l e u r o t u s , we a l s o

l o o k e d f o r d i r e c t or i n d i r e c t c o r r e l a t i o n s between s u b s t r a t e and spo r e d i m e n s i o n , a q u e s t i o n o f g e n e r a l i m p o r t a n c e . A l t h o u g h a t the moment o n l y o f p r e l i m i n a r y n a t u r e , our d a t a , o b t a i n e d from a w e l l s u i t e d o r g a n i s m , a l l o w us t o t a k e up a g a i n the d i s c u s s i o n o f sub­s t r a t e m o d i f i c a t i o n of s p o r e s , a problem which i s i m p o r t a n t e s p e c i a l ­l y i n l o w e r p a r a s i t i c f u n g i .

F r u i t b o d i e s of one s i n g l e s t r a i n o f P_. o s t r e a t u s ( s t r a i n 2y) were grown on d i f f e r e n t s u b s t r a t e s and t h e d i s t r i b u t i o n of sp o r e dimen­s i o n s was d e t e r m i n e d . The l e n g t h of s p o r e s showed g r e a t e r v a r i a t i o n t h a n t h e d i a m e t e r o f s p o r e s and was t h e r e f o r e used f o r p r e s e n t a t i o n o f r e s u l t s ( F i g . 3 ) . 100 s p o r e s each f o r ever y e x p e r i m e n t were measu­r e d . G r e a t e s t v a r i a t i o n i n s p o r e l e n g t h was found i n the f r u i t b o d i e s from P_. o s t r e a t u s on S a l i x , t h e maximum o f f r e q u e n c y b e i n g around 9,5 m i c r o n s ; t h e s h o u l d e r around 11 m i c r o n s c o u l d r e p r e s e n t a f u r t h e r , h i d d e n maximum. UJith e x c e p t i o n o f t h e f r u i t b o d i e s on stumps o f P i -cea, i n a l l o t h e r e x p e r i m e n t s v a r i a t i o n of spore d i a m e t e r s was n a r r o ­wer. The av e r a g e l e n g t h of s p o r e s from wheat s t r a w was s m a l l e r , t he peak o f f r e q u e n c y b e e i n g around 8,5 m i c r o n s . C l e a r l y h i g h e r were t h e v a l u e s f o r s p o r e s from f r u i t b o d i e s on stumps of P i c e a where t h e ma­ximum f r e q u e n c y was a t 11,5 m i c r o n s . D i f f e r e n t s u b s t r a t e s t h e r e f o r e have a s t r o n g i n f l u e n c e on t h e average d i m e n s i o n o f s p o r e s (8.5 -10.95 m i c r o n s ) and the maxima o f f r e q u e n c y may be i n extreme c a s e s as f a r a p a r t as 3 m i c r o n s .

A f i n a l i n t e r p r e t a t i o n of t h e r e s u l t s cannot be g i v e n a t t h e moment. The d i f f e r e n t s u b s t r a t e s e i t h e r c o u l d modify d i r e c t l y or a c t i n d i ­r e c t l y on t h e d i c a r y o n s t h r o u g h s e l e c t i o n , r e s u l t i n g i n t h e o b s e r v e d p i c t u r e .

From a l l t h i s f o l l o w s t h a t t a x o n o m i c c h a r a c t e r s may e x h i b i t c o n s i d e ­r a b l e f l u c t a t i o n due t o d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s . However, the a v e r a g e e n v i r o n m e n t a l s i t u a t i o n which i n d u c e s f r u i t i n g i n the f i e l d l i m i t s t h e range of p h e n e t i c p l a s t i c i t y . Thus o v e r l a p p i n g o f f e a t u r e s due t o un e q u a l s h i f t of e n v i r o n m e n t a l c o n d i t i o n s as p o s s i b l e i n t h e l a b o r a t o r y s h o u l d not be o v e r e m p h a s i z e d i n r e g a r d t o the prob­lem o f f i n d i n g p r o p e r p h e n e t i c d i f f e r e n c e s f o r the d e l i m i t a t i o n of s p e c i e s .

4. I n t e r s t e r i l i t y b a r r i e r s b e t w e e n s p e c i e s o f P l e u r o t u s

In the f o l l o w i n g two c h a p t e r s i n a b i l i t y o f gene exchange between d i f ­f e r e n t s p e c i e s i s c o n t r a s t e d w i t h i n t e r n a l f e r t i l i t y w i t h i n s p e c i e s . Lack of e x t e r n a l i n t e r b r e e d i n g combined w i t h the p r e s e n c e of morpho­l o g i c a l d i f f e r e n c e s between the u n i t s s u p p o r t s t h e v a l i d i t y o f an adopted s p e c i e s c o n c e p t . R e g a r d i n g the "bona f i d e " - s p e c i e s of P l e u ­r o t u s t r e a t e d i n the key t h i s means n e c e s s i t y of v a l i d a t i o n by l o o ­k i n g f o r the e s t a b l i s h e d m a t i n g b a r r i e r s . T h i s p r o c e d u r e was f o l l o w e d i n some o f the p o s s i b l e c o m b i n a t i o n s .

a) P_. p u l m o n a r i u s x P_ . c o l u m b i n u s For both s p e c i e s (P. c o l u m b i n u s , s t r a i n I n ; P. pulmonar i u s j i s t r a i n l r ) the m a t i n g t y p e s were d e t e r m i n e d and the e x p e c t e d scheme of t e t r a p o -l a r i n c o m p a t i b i l i t y has been c o n f i r m e d . The m o n o c a r y o t i c m y c e l i a were o b t a i n e d from a s i n g l e s p o r o c a r p of each of both s p e c i e s . For each s p e c i e s m a t i n g s of the m o n o c a r y o t i c m y c e l i a r e s u l t e d i n 25/£ f o r m a t i o n of clamp c o n n e c t i o n s ( t a b l e l a and l b ) . However, i n no case clamp c o n n e c t i o n s o c c u r r e d i n c o n f r o n t a t i o n s between m o n o c a r y o t i c m y c e l i a of P_. p u l m o n a r i u s and £. c o l u m b i n u s ( t a b l e l c ) . U s i n g d i f f e r e n t s t r a i n s of both s p e c i e s the c r o s s e s were r e p e a t e d w i t h the same r e ­s u l t : i n no case f o r m a t i o n of clamp c o n n e c t i o n s c o u l d be o b s e r v e d .

b) £ . p u l m o n a r i u s x £ . o s t r e a t u s Not any c o m b i n a t i o n of monocaryons of t h e s e two s p e c i e s l e d t o clamp f o r m i n g m y c e l i a ( i d x l u and l r x I s ) .

c) £ . p u l m o n a r i u s x £ . c o r n u c o p i a e C r o s s e s of monocaryons of P_. p u l m o n a r i u s ( s t r a i n l r ) w i t h monocaryons of £. c o r n u c o p i a e ( s t r a i n 4 r ) d i d not y i e l d any clamp c o n n e c t i o n s . From t h e s e o b s e r v a t i o n s f o l l o w s t h a t £. p u l m o n a r i u s r e p r e s e n t s an i n ­dependent s p e c i e s , which i s g e n e t i c a l l y i s o l a t e d from £. co l u m b i n u s and £. o s t r e a t u s i n s p i t e of o n l y minute d i f f e r e n c e s i n m a c r o s c o p i c a l and i n m i c r o s c o p i c a l f e a t u r e s . f u r t h e r i n t e r s p e c i e s c r o s s e s a r e l i s t e d i n t a b l e 2.

5. I n t e r n a l f e r t i l i t y w i t h i n t h e s p e c i e s o f P l e u r o t u s

A c c o r d i n g t o m u l t i p l e a l l e l i s m o f i n c o m p a t i b i l i t y genes the p e r c e n ­t a g e of d i c a r y o t i z a t i o n between s t r a i n s of one s p e c i e s i n c r e a s e s at a c o n s i d e r a b l e r a t e i f t h e s e s t r a i n s a re of d i f f e r e n t o r i g i n . T h i s phe­nomenon i s d e m o n s t r a t e d w i t h d i f f e r e n t s t r a i n s of _P_. p u l m o n a r i u s and P_. o s t r e a t u s ( t a b l e 3 a-3d). D i c a r y o t i z a t i o n o c c u r r e d between s t r a i n s which showed c l e a r d e v i a t i o n of s e v e r a l p r o p e r t i e s . S i n c e r e c o m b i n a ­t i o n o f v a r y i n g c h a r a c t e r s i s f a v o r e d by m u l t i p l e a l l e l i s m o f incom­p a t i b i l i t y genes t h i s means i n c r e a s e of i n f r a s p e c i f i c v a r i a t i o n . New s p e c i e s may p r e d o m i n a n t l y o r i g i n a t e from such u n i t s w i t h a h i g h de­gree o f i n t e r n a l v a r i a t i o n .

C r o s s e s between monocaryons w i t h t h r o u g h o u t d i f f e r e n t A- and B - f a c -t o r s p o s s e s s a c c o r d i n g t o E ugenio and Anderson (1968) an i n c r e a s e d chance t o s u c c e e d , s i n c e t h e t o t a l w e i g h t of f r u i t b o d i e s i n c r e a s e s and by t h a t t h e number of s p o r e s i s a l s o h i g h e r .

Some remarks on t h e r e s u l t s p r e s e n t e d i n t a b l e 3 a r e n e c e s s a r y i n o r ­der t o d e m o n s t r a t e exchange o f d i f f e r i n g f e a t u r e s i n i n t r a s p e c i e s c r o s s e s . The f r u i t b o d i e s of P_. p u l m o n a r i u s 4h a r e l i g h t e r ( n e a r l y w h i t e ) t h a n t h o s e of P. p u l m o n a r i u s l r ( t a b l e 3b: l r x 4 h ) .

The s o - c a l l e d s t r a i n " F l o r i d a " , o r i g i n a l l y o b t a i n e d by Eger from F l o r i d a , became i m p o r t a n t f o r c o m m e r c i a l c u l t i v a t i o n , because i t does not need a c o l d - p h a s e f o r i n d u c t i o n of f r u i t i n g . The f a c t t h a t t h i s s t r a i n " F l o r i d a " i n t e r b r e e d s w i t h P. p u l m o n a r i u s l r from A u s t r i a shows t h a t t h e former cannot been t a k e n as an i n d e p e n d a n t s p e c i e s ( t a b l e 3c: l r x 4 b ) . E g e r , Eden and U J i s s i g (1976) d e s c r i b e t h e ma­t i n g o f a s t r a i n from F l o r i d a w i t h some s t r a i n s from A m e r i c a and Germany. The m o n o c a r y o t i c m y c e l i a of t h e s e s t r a i n s were i n t h e c r o s ­s i n g s 100% c o m p a t i b l e and l a r g e l y f e r t i l e ( r e g a r d i n g p r o d u c t i o n of h y b r i d s p o r o c a r p s and s p o r e s ) . U n f o r t u n a t e l y i t i s i m p o s s i b l e t o i d e n t i f y t h e s e s t r a i n s from Germany used by Eger et a l . by t h e g i v e n g e n e r a l remarks about c o l o r changes due t o d i f f e r e n t l i g h t i n t e n s i ­t i e s . The a s s u m p t i o n of t h e s e a u t h o r s , h a v i n g c r o s s e d P l e u r o t u s " s t r a i n F l o r i d a " w i t h P_. o s t r e a t u s , t h e r e f o r e cannot be v e r i f i e d . For our p r o b l e m , however, i t i s i m p o r t a n t t h a t Eger e t a l . were a b l e t o prpve t h a t i n t e r b r e e d i n g was l i n k e d w i t h c o m b i n a t i o n o f d i f f e r e n t

p r o p e r t i e s , e.g. t e m p e r a t u r e o p t i m a of f r u i t i n g .

M o n o c a r y o t i c i s o l a t e s of P_. o s t r e a t u s from Germany, U J e s t f a l e n , a r e 100/6 c o m p a t i b l e w i t h two d i f f e r e n t s t r a i n s o f £. o s t r e a t u s from Japan ( t a b l e 3d: I s x l w ; I s x l u ) . R e c o m b i n a t i o n t a k e s p l a c e between s t r a i n s which d i f f e r i n t h e c o l o r of the cap. The c o l o r s of the f u n g i from Japan a r e l i g h t e r , e s p e c i a l l y i n o l d e r s t a g e s , t h a n t h o s e o f t h e s t r a i n o b t a i n e d from U J e s t f a l e n . The F i - o f f s p r i n g of h y b r i d f r u i t bo­d i e s , which were f e r t i l e i n r e g a r d t o s p o r e p r o d u c t i o n , was s p l i t i n ­t o d a r k e r and l i g h t e r c o l o r e d i n d i v i d u a l s .

As a r e s u l t o f t h e s e o b s e r v a t i o n s we may s t a t e , t h a t d i f f e r e n c e s i n f e a t u r e s between two compared u n i t s a r e not per se r e l i a b l e c r i t e r i a t o e s t a b l i s h a s p e c i e s c o n c e p t . In e v e r y case i t i s n e c e s s a r y t o be su r e t h a t t h e d i f f e r e n c e s a r e m a i n t a i n e d by p r o p e r i s o l a t i o n mecha­nisms .

6. E n z y m e s p e c t r a f o r c h a r a c t e r i z a ­t i o n o f s p e c i e s a n d s t r a i n s o f P l e u r o t u s

C h e m i c a l a n a l y s i s as t o o l of " b i o c h e m i c a l s y s t e m a t i c s " (Heywood, 1973a) has been used s u c c e s s f u l l y and i n c r e a s i n g l y w i t h i n the l a s t y e a r s i n s y s t e m a t i c s and taxonomy of h i g h e r p l a n t s and f u n g i ( D a v i s and Heywood, 1963; H a l l , 1973; Vaughan, 1975; but see Heywood, 1973b f o r c r i t i c a l comments).

P r o t e i n s a r e - a c c o r d i n g t o the one-gene-one-enzyme-hypotheses ( B e a d l e and Tatum, 1941) - d i r e c t p r o d u c t s of genes and t h e r e f o r e a l ­so i n d i c a t i v e f o r the g e n e t i c m a t e r i a l . For t h a t r e a s o n p r o t e i n s , t o ­g e t h e r w i t h p i g m e n t s , have been a n a l y z e d f o r p r e s e n c e , q u a l i t y and d i s t r i b u t i o n by e l e c t r o p h o r e t i c a l and i n the case of p r o t e i n s a l s o s e r o l o g i c a l methods. A number of good c o r r e l a t i o n s w i t h m o r p h o l o g i c a l and o t h e r d a t a has been found (see H a l l , 1969; T y r e l l , 1969; H a l l , 1973; Bucher, 1974).

G e n e r a l p r o t e i n p r o f i l e s a r e o f t e n o b s c u r e d and u n s p e c i f i c by too ma­ny p r o t e i n b a n d s . I t t h e r e f o r e p r oved advantageous e i t h e r o n l y t o i n ­v e s t i g a t e c e r t a i n f r a c t i o n s of t o t a l p r o t e i n or - even b e t t e r - o n l y s p e c i f i c enzymes, c h a r a c t e r i s t i c f o r a g i v e n o r g a n i s m . UUith due c a r e

• f i n t e r p r e t a t i o n such isoenzyme s p e c t r a appear t o be u s e f u l f o r t a ­xonomic and s y s t e m a t i c p u r p o s e s a l s o f o r f u n g i ( N e a l s o n and G a r b e r , 1967; Wang and Raper, 1969; Reddy and T h r e l k e l d , 1971; Snyder and Kramer, 1 9 7 4 ) .

For t h e s e r e a s o n s i t seemed t o be w o r t h w h i l e w i t h i n t h e scope of our problem t o i n v e s t i g a t e not o n l y t h e m o r p h o l o g i c c r i t e r i a o f t h e wood-r o t t i n g genus P l e u r o t u s ( w h i t e r o t ) but a l s o t h e enzymes b e l i e v e d t o be r e s p o n s i b l e f o r t h e decay of wood. We t e s t e d t h e r e f o r e the pheno-l o x i d a s e s t y r o s i n a s e and l a c c a s e and i n a d d i t i o n p e r o x i d a s e . I t was hoped t o f i n d o u t , whether enzyme p r o f i l e s i n a d d i t i o n t o t h e e s t a b ­l i s h e d m o r p h o l o g i c a l c r i t e r i a c o u l d p r o v i d e u s e f u l i n f o r m a t i o n f o r the d e l i m i t a t i o n of s p e c i e s and c h a r a c t e r i z a t i o n o f s t r a i n s i n f u n g i .

For t h i s p urpose s e v e r a l s t r a i n s of P l e u r o t u s (P_. c o l u m b i n u s , P_. os­t r e a t u s , P_. p u l m o n a r i u s ) were t e s t e d . I n o r d e r t o see the e f f e c t o f t h e n u c l e a r s t a t u s on growth and enzyme s p e c t r a , we f u r t h e r m o r e ana­l y z e d o f one s p e c i e s , P_. o s t r e a t u s , m o n o c a r y o t i c s t r a i n s ( l w 8 , l s 4 , d i c a r y o t i c s t r a i n s ( l w , I s ) and t h e d i c a r y o t i c h y b r i d o f t h e c r o s s lw x I s .

At f i r s t g r o w t h r a t e and y i e l d o f m y c e ­l i u m on d i f f e r e n t s o l i d and l i q u i d media a t d i f f e r e n t tempera­t u r e s were d e t e r m i n e d i n o r d e r t o c h a r a c t e r i z e t h e s t r a i n s and t o ob­t a i n t h e o p t i m a l t ime of h a r v e s t f o r the enzyme d e t e r m i n a t i o n s . T a b l e 4 p r e s e n t s t h e r e s u l t s f o r growth on m a l t - a g a r and m a l t - b r o t h a t 27°C. The P l e u r o t u s s p e c i e s and s t r a i n s showed c h a r a c t e r i s t i c and c o n s t a n t growth r a t e s on m a l t - a g a r , the growth r a t e s b e i n g i n t h e range o f v a l u e s g i v e n by N o b l e s ( 1 9 6 5 ) , (Ylacaya - L i z a n o (1974-1975) and B l a i c h ( 1 9 7 2 ) . Only s m a l l d i f f e r e n c e s i n growth r a t e e x i s t e d b e t ­ween the s t r a i n s on s o l i d media, whereas i n l i q u i d medium m y c e l i a l y i e l d showed w i d e r v a r i a t i o n . I t i s s t r i k i n g t h a t on s o l i d medium t h e growth r a t e o f t h e h y b r i d lw x I s i s about t w i c e as h i g h as t h a t o f the m o n o c a r y o t i c c r o s s i n g p a r t n e r s lw8 and l s 4 , whereas i n l i q u i d me­dium m y c e l i a l y i e l d of t h e h y b r i d i s o n l y about one q u a r t e r o f t h a t of t h e m o n o c a r y o t i c s t r a i n s .

L o o k i n g at t h e e n z y m e a c t i v i t i e s i n s o l i d and l i ­q u i d medium, p e r o x i d a s e c o u l d not be found i n any s t r a i n w i t h t h e me­t h o d s u s e d , n e i t h e r on s o l i d medium nor c o l o r i m e t r i c a l l y i n c u l t u r e

f i l t r a t e s and m y c e l i a l e x t r a c t s nor by s t a i n i n g of g e l s a f t e r e l e c ­t r o p h o r e s i s . T h i s c o n f i r m s t h e r e s u l t s o f L y r (1958) f o r o s t r e a ­t u s .

A f t e r 2 weeks o f i n c u b a t i o n t y r o s i n a s e a l s o was a b s e n t i n m a l t - a g a r , i n c u l t u r e f i l t r a t e s and i n m y c e l i a l e x t r a c t s o f l i q u i d c u l t u r e . T h i s enzyme a p p e a r e d , however, i n s m a l l amounts a f t e r 3 weeks on s o l i d me­dium i n c u l t u r e s of P_. c o l u m b i n u s and P_. o s t r e a t u s . Only t h e monoca­r y o t i c s t r a i n s and t h e d i c a r y o t i c h y b r i d o f P. o s t r e a t u s d i d not show t y r o s i n a s e a c t i v i t y a t t h i s t i m e . P r e s e n c e of t y r o s i n a s e i n P_. colum­b i n u s and _P. o s t r e a t u s a g r e e s w i t h t h e d a t a o f H a c k l ( 1 9 7 5 ) . Our r e ­s u l t s show f u r t h e r t h e dependency of t y r o s i n a s e p r o d u c t i o n from c u l ­t u r a l age.

L a c c a s e was a l w a y s found i n every s t r a i n t e s t e d i n s o l i d media w i t h t h e d r o p - t e s t ( e x t r a c e l l u l a r enzyme), i n t h e c u l t u r e f i l t r a t e s ( ex­t r a c e l l u l a r enzyme) and i n m y c e l i a l e x t r a c t s ( i n t r a c e l l u l a r enzyme) from l i q u i d c u l t u r e . T h i s c o r r e s p o n d s w e l l w i t h t h e d a t a o f L y r ( 1 9 5 8 ) , H a r k i n e t a l . ( 1 9 7 4 ) , B l a i c h and E s s e r ( 1 9 7 5 ) , H a c k l (1975) and Leonowicz and T r o j a n o w s k i (1975a, 1975b).

The d i s c r e p a n c y between the s e m i q u a n t i t a t i v e d a t a o f t h e d r o p - t e s t and t h e q u a n t i t a t i v e c o l o r i m e t r i c d e t e r m i n a t i o n s of l a c c a s e a c t i v i t y s u g g e s t s t h a t t h e MBavendamm-test M ( L y r , 1958) s h o u l d be used o n l y f o r q u a l i t a t i v e o r p r e l i m i n a r y d e t e r m i n a t i o n s and shows t h a t t h i s t e s t i n d i c a t e s o n l y p r e s e n c e o f e x t r a c e l l u l a r enzymes.

In o r d e r t o check a l s o t h e p r e s e n c e of i n t r a c e l l u l a r enzymes m y c e l i a l e x t r a c t s i n a d d i t i o n t o t h e c u l t u r e f i l t r a t e s were i n v e s t i g a t e d . From t a b l e 4 a p p e a r s t h a t l a c c a s e i s here p r e d o m i n a n t l y an e x t r a c e l l u l a r enzyme. The monocaryons o f P_. o s t r e a t u s s y n t h e s i z e o n l y about l / 3 o f t h e l a c c a s e produced by the d i c a r y o n s and the h y b r i d . The i n v e r s e r e ­l a t i o n s h i p between p h e n o l o x i d a s e - and mycelium p r o d u c t i o n (see above) has been o b s e r v e d r e p e a t e d l y (Bergmann, ( Y l o l i t o r i s , u n p u b l i s h e d r e ­s u l t s ) .

As c o u l d be shown, a more s p e c i f i c and b e t t e r r e p r o d u c i b l e p i c t u r e of the e n z y m a t i c d i f f e r e n c e s between the s t r a i n s was o b t a i n e d from t h e isoenzyme s p e c t r a o f d i s c - e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g .

As f i g u r e 5 shows, i n t h e c u l t u r e f i l t r a t e s g e n e r a l l y fewer l a c c a s e bands were f o u n d , an i n d i c a t i o n f o r p r e v e n t i o n or r e t a r d a t i o n o f l a c ­case s e c r e t i o n t h r o u g h the c e l l membrane i n t o the medium ( [ Y l o l i t o r i s and E s s e r , 1 9 7 1 ) .

In d i s c - e l e c t r o p h o r e s i s a t pH 8.3 i n a l l c a s e s a l a c c a s e band remains i n the s p a c e r g e l , i n d i c a t i n g a l a c c a s e of h i g h i s o e l e c t r i c p o i n t l i k e t he B-type l a c c a s e s w i t h an i s o e l e c t r i c p o i n t between pH 4 t o 8 ( J o n s s o n e t a l . , 1968) or l a c c a s e I I o f Podospora a n s e r i n a ( ( Y l o l i t o r i s and E s s e r , 1971). In a d d i t i o n , a l l s t r a i n s show a s t r o n g l a c c a s e band near t h e moving boundary, i n d i c a t i n g a l a c c a s e o f low m o l e c u l a r w e i g h t and/or low i s o e l e c t r i c p o i n t c o r r e s p o n d i n g w i t h the A-type l a c c a s e s o f J o n s s o n e t a l . ( 1 9 6 8 ) . A d d i t i o n a l bands appea­r e d i n the m y c e l i a l e x t r a c t s o f _P_. c o l u m b i n u s and P. o s t r e a t u s and showed c h a r a c t e r i s t i c d i f f e r e n c e s between the s t r a i n s . In P_. o s t r e a ­t u s such a d d i t i o n a l bands were found o n l y i n t h e m y c e l i a l e x t r a c t s of the mono- and d i c a r y o t i c " s " - s t r a i n s ( l s 4 and I s ) but not i n t h e mono- and d i c a r y o t i c " w " - s t r a i n s (lw7 and lw) nor i n t h e h y b r i d lw x I s . T h i s h y b r i d s t r a i n t h e r e f o r e i s n o t , as c o u l d be e x p e c t e d , i n t e r m e d i a t e i n i t s l a c c a s e spectrum but i s i d e n t i c a l w i t h one of the p a r e n t a l s t r a i n s ( l w 8 ) .

I s o e l e c t r i c f o c u s i n g g i v e s c o n s i d e r a b l y b e t t e r s e p a r a t i o n t h a n d i s c e l e c t r o p h o r e s i s . I t i s t h e r e f o r e not s u r p r i s i n g t h a t t h i s method produced more d i s t i n c t l a c c a s e bands. The danger of a r t i f a c t s , however, i s i n i s o e l e c t r i c f o c u s i n g h i g h e r . A g a i n , A- and B-type l a c c a s e s a r e p r e s e n t and bo t h a r e s e c r e t e d i n t o t he c u l t u r e medium. A l l s p e c i e s and s t r a i n s of P l e u r o t u s showed d i f f e r e n t l a c c a s e s p e c t r a . The s i m i l a r i t y o f l a c c a s e s p e c t r a i n i s o e l e c t r i c f o c u s i n g w i t h i n a s p e c i e s was g e n e r a l l y not h i g h e r than between s p e c i e s . An e x c e p t i o n a r e t h e s p e c t r a o f the c u l t u r e f i l t r a t e s o f _P_. o s t r e a t u s . In t h e case o f t h e s p e c t r a from i s o e l e c t r i c f o c u s i n g i n t e r p r e t a t i o n of r e s u l t s s h o u l d a w a i t f u r t h e r e x p e r i m e n t a t i o n .

As a r e s u l t o f t h e s e p r e l i m i n a r y i n v e s t i g a t i o n s i t can be s t a t e d a l ­r e a d y : The a n a l y z e d s t r a i n s o f P l e u r o t u s a l l produce i n t r a - and ex­t r a c e l l u l a r l a c c a s e and e x h i b i t c h a r a c t e r i s t i c , r e p r o d u c i b l e i s o e n ­zyme s p e c t r a i n e l e c t r o p h o r e s i s . The s p e c t r a o f the h y b r i d (lw x I s ) o f _P_. o s t r e a t u s show i n d i s c - e l e c t r o p h o r e s i s i d e n t i t y w i t h one o f the c r o s s i n g p a r t n e r s ( l w ) , i n i s o e l e c t r i c f o c u s i n g the s p e c t r a o f the

c u l t u r e f i l t r a t e are a t l e a s t v e r y s i m i l a r t o both m o n o c a r y o t i c c r o s ­s i n g p a r t n e r s .

B e f o r e tue f i n a l l y can a s s e s s the s u i t a b i l i t y of t h e s e methods f o r c h a r a c t e r i z a t i o n of s t r a i n s and s p e c i e s of P l e u r o t u s a number of ad­d i t i o n a l enzymes s h o u l d be t e s t e d as w e l l as the i n f l u e n c e on the en­zyme s p e c t r a o f f a c t o r s l i k e m y c e l i a l age, medium and e n v i r o n m e n t a l c o n d i t i o n s l i k e t e m p e r a t u r e and l i g h t .

7. S p e c i e s c o n c e p t A s p e c i e s c o m p r i s e s a l l i n d i v i d u a l s w i t h i n d e n t i c a l or c o n t i n o u s l y v a r y i n g c h a r a c t e r s . I n d i v i d u a l s b e l o n g t o d i f f e r e n t s p e c i e s i f the v a r i a t i o n o f c h a r a c t e r s shows d i s c o n t i n u i t i e s . S p e c i e s a r e r e c o g n i z e d and d e f i n e d by such d i s c o n t i n u i t i e s ( D a v i s and Heywood, 1963). A p r o ­per d e l i m i t a t i o n of s p e c i e s i s not i n f l u e n c e d by the q u e s t i o n how ma­ny genes p a r t i c i p a t e i n an e s t a b l i s h e d d i s c o n t i n u i t y i f s t a b i l i t y o f d i f f e r e n c e s i s w a r r a n t e d on one s i d e , and c o n t i n u i t y o f v a r i a t i o n i s m a i n t a i n e d w i t h i n t h e d e f i n e d s p e c i e s on the o t h e r s i d e . G e n e t i c i s o ­l a t i o n i s t h e most i m p o r t a n t mechanism f o r e s t a b l i s h i n g d i s c o n t i n u i ­t i e s i n t h e v a r i a t i o n o f f u n g i .

In o r d e r t o l i m i t a t e t a x o n o m i c work w i t h b a s i d i o m y c e t e s on the spe­c i e s l e v e l t h e s p e c i e s c oncept s h o u l d be based p r i m a r i l y on macro-and m i c r o s c o p i c , macro- and m i c r o c h e m i c a l and f i n a l l y on e c o l o g i c a l c r i t e r i a . P h y s i o l o g i c a l and b i o c h e m i c a l c h a r a c t e r s would o n l y be use­f u l i f the i n b r e e d i n g u n i t s ( i . e . t h e s p e c i e s ) a r e c h a r a c t e r i z e d as a whole by such c r i t e r i a . O t h e r w i s e t h e s e f e a t u r e s would o n l y s e r v e t o d e s c r i b e the i n t e r n a l v a r i a t i o n and s t r u c t u r e of s p e c i e s .

The s p e c i e s c oncept f o r t h e b a s i d i o m y c e t e s seems t o be c o m p r e h e n s i b l e i n an a l m o s t o b j e c t i v e manner, a l t h o u g h t h e r e a r e d i f f i c u l t i e s i n ap­p l i c a t i o n . I n many groups g e n e t i c i s o l a t i o n cannot be a s s a y e d by ma­t i n g e x p e r i m e n t s , and taxonomy o f b a s i d i o m y c e t e s i s t h e r e f o r e based on a " b o n a - f i d e " - s p e c i e s c o n c e p t . E n v i r o n m e n t a l c o n d i t i o n s may i n ­f l u e n c e f e a t u r e s t o a h i g h degree and t h i s i n h i b i t s p r e c i s e d e t e r m i ­n a t i o n o f s p e c i f i c d i f f e r e n c e s .

R e c o g n i t i o n o f d i s c o n t i n u i t i e s and t h e r e b y o f s p e c i e s i s based on l o n a e x p e r i e n c e , much p a t i e n c e and on t h e o b s e r v a t i o n o f o f t e n minute

d i f f e r e n c e s . T h i s demands a p t i t u d e f o r c a r r y i n g out such o b s e r v a ­t i o n s , w h i ch i s not g i v e n e v e r y o n e . F i n a l l y , s p e c i e s a r e dynamic s t r u c t u r e s which can be a l t e r e d by e v o l u t i o n a r y p r o c e s s e s . To r e ­c o g n i z e such changes i s f o r the s y s t e m a t i s t - c o n t r a r y t o common T i e w - not annoyance but o b j e c t o f h i s s t u d i e s .

fl. M a t e r i a l a n d m e t h o d s J w s o l a t e s , pure c u l t u r e s and m o n o c a r y o t i c m y c e l i a mere o b t a i n e d and p r o p a g a t e d u s i n g c o n v e n t i o n a l methods.

f r u i t b o d i e s , v e r y s i m i l a r t o t h o s e grown i n n a t u r e , were produced by f i l l i n g E r l e n m e y e r - f l a s k s (300 m l , wide mouth) h a l f -,*oay w i t h wheat g r a i n . A f t e r a d d i t i o n o f d i s t i l l e d water the g r a i n s w e l l e d and f i l l e d t h e f l a s k s up t o the r i m . The f l a s k s t h e n were i n o c u l a t e d , c l o s e d w i t h c o t t o n p l u g s , c o v e r e d w i t h a l u m i n i u m f o i l and i n c u b a t e d a t 23°C. A f t e r 14 days the f l a s k s were t r a n s f e r r e d t o 11°C and 500 - 600 l u x c o n s t a n t l i g h t . The f o i l was removed on one s i d e t o a l l o w l a t e r a l growth o f the f u n g u s , s i m u l a t i n g n a t u r a l c o n d i t i o n s . F r u i t b o d i e s were produced a f t e r 4 (3 - 6) weeks, depending on s t r a i n and s p e c i e s . T h i s method was s u p e r i o r t o f r u i t body p r o d u c t i o n i n s i d e f l a s k s and p e t r i - d i s h e s .

F l a s k s : The f l a s k s were f i l l e d w i t h 30 g o f s u b s t r a t e : a) s t r a w o f wheat; sawdust o f b) Fagus, c) P i c e a , d) A b i e s , 3) B e t u l a . The s u b s t r a t e was c o v e r e d by a t h i n f i l m of the f o l l o w i n g c o m p o s i t i o n : I n o s i t o l 50 mg; KH2PO4 0,5 g; lYlgSO^ 0,5 g; ZnSO^ 0,001g, F e C l 3 0,01 g, C a C l 2 0,055 g, fYln50 4 0,005 g, m a l t o s e 20 g, g l u c o s e 10 g, a g a r - a g a r 20 g, a s p a r a g i n e 1.2 g, a l a n i n e 0,8 g, d e s t . w a t e r ad 1000 ml.

The f l a s k s were i n o c u l a t e d and i n c u b a t e d f o r 14 days i n the dark a t 23°C, t h e n t r a n s f e r r e d t o 11°C and 500 - 600 l u x l i g h t i n t e n s i t y ; r e ­moval of the p l u g , 80^ r e l a t i v e a i r h u m i d i t y . F r u i t b o d i e s were p r o ­duced a f t e r 8 (6 - 10) weeks.

P e t r i - d i s h e s : 20 g of s u b s t r a t e , c o v e r e d w i t h a f i l m o f medium as above. The p e t r i - d i s h e s were kept c l o s e d i n the f i r s t i n c u ­b a t i o n p e r i o d . D u r i n g t h e ll°C-period they were opened and kept under a t e n t o f pol y e t h y l e n e f o i l . CO2, i n h i b i t i n g f r u i t i n g body p r o d u c t i o n ,

uas removed by a b s o r p t i o n by a s o l u t i o n of Ca(0H ) 2 .

M y c e l i a l g r o w t h : Growth i n c o n n e c t i o n w i t h t h e enzyme d e t e r m i n a t i o n s was o b s e r v e d on m a l t - b r o t h ( 1 , 5 ^ m a l t ) , s o l i d i f i e d i f needed by 1,5% a g a r . The l i q u i d c u l t u r e s ( s u r f a c e c u l t u r e , 250 ml E r l e n m e y e r - f l a s k s c o n t a i n i n g 90 ml o f medium) were i n o c u l a t e d w i t h 10 ml o f a homogenate of a 14 day o l d l i q u i d p r e - c u l t u r e . A f t e r 14 days of i n c u b a t i o n a t 27°C and d i f f u s e l i g h t , the mycelium was h a r ­v e s t e d and s e p a r a t e d from the c u l t u r e f i l t r a t e by s u c t i o n t h r o u g h f i l t e r p a p e r on a B u c h n e r - f u n n e l . Wet w e i g h t , dry w e i g t h (80°C) and pH were d e t e r m i n e d f o r each e x p e r i m e n t .

C u l t u r a l f i l t r a t e a n d m y c e l i a l e x ­t r a c t s . 5 g mycelium (wet w e i g h t ) were homogenized f o r 3 min i n a p r e c o o l e d m o r t a r w i t h 10 g of washed seasand. A f t e r a d d i t i o n of 30 ml 0,05 M phosphate b u f f e r , pH 6.0, t h e mycelium was ground f o r a n o t h e r 5 min. M y c e l i a l f r a g m e n t s were s e p a r a t e d from the m y c e l i a l e x t r a c t by c e n t r i f u g a t i o n (20 min, 4°C, 10,000 x g ) . I f n e c e s s a r y , m y c e l i a l e x t r a c t s and c u l t u r a l f i l t r a t e s were c o n c e n t r a t e d f o r the enzyme a s s a y s i n d i s c - e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g by u l t r a f i l t r a t i o n .

E l e c t r o p h o r e t i c s e p a r a t i o n o f p r o ­t e i n s : D i s c - e l e c t r o p h o r e s i s was c o n d u cted a c c o r d i n g t o O r n s t e i n and D a v i s (1964) at pH 8,3 i n 7,5^ p o l y a e r y l a m i d e g e l , i s o e l e c t r i c f o c u s i n g i n a p H - g r a d i e n t from pH 3,5 t o 10 i n 7,5^ p o l y a c r y l a m i d e g e l a c c o r d i n g t o W r i g l e y ( 1 9 6 8 ) .

E n z y m e a s s a y s : P r e s e n c e of p e r o x i d a s e on m a l t - a g a r was d e t e r m i n e d a f t e r L y r ( d r o p - t e s t , 1958) w i t h a s o l u t i o n o f 0.1% b e n z i ­d i n e i n e x t r a c t s and c u l t u r a l f i l t r a t e s p e r o x i d a s e was a s s a y e d f o r by a c o l o r i m e t r i c t e s t a t 436 nm w i t h g u a j a c o l and H2O2 a c c o r d i n g t o P u t t e r ( 1 9 7 4 a , b ) .

T y r o s i n a s e on m a l t - a g a r was d e t e r m i n e d f o l l o w i n g L y r ( 1 9 5 8 ) , u s i n g an aqueous s o l u t i o n of 0,01 M p - c r e s o l c o n t a i n i n g 0,05 g l y c i n e . T y r o s i n a s e i n c u l t u r a l f i l t r a t e s and m y c e l i a l e x t r a c t s was t e s t e d c o l o r i m e t r i c a l l y a t 436 mm w i t h 0,02 M L - t y r o s i n e ( E s s e r , 1963).

L a c c a s e on m a l t - a g a r was a s s a y e d f o r a c c o r d i n g t o L y r (1958) w i t h an aqueous s o l u t i o n o f 0,005^ a - n a p h t h o l or 0,005^ g u a i a c o l . P r e s e n c e o f l a c c a s e i n c u l t u r e d f i l t r a t e s or m y c e l i a l e x t r a c t s was i n v e s t i g a t e d c o l o r i m e t r i c a l l y a t 436 nm w i t h 0,65mM 2,6-dimethoxyphe­n o l i n 0,1IY1 N a - c i t r a t e / N a O H b u f f e r , pH 5,0 a c c o r d i n g t o P r i l l i n g e r ( 1 9 7 6 ) . L a c c a s e i n t h e e l e c t r o p h o r e t i c e x p e r i m e n t s was d e t e r m i n e d by i n c u b a t i o n o f t h e g e l s i n 0.02 IY1 g u a i a c o l i n 0.05 IYI phosphate b u f f e r , pH 6.0. The g e l s a f t e r i s o e l e c t r i c f o c u s i n g were e q u i l i b r a t e d i n b u f ­f e r f o r 30 min b e f o r e a d d i t i o n o f the s u b s t r a t e .

1 u n i t of l a c c a s e - a c t i v i t y ( I E ) c o r r e s p o n d s w i t h a d i f f e r e n c e i n ab­s o r p t i o n o f 0.2/min a t 436 nm and 1 cm l i g h t p a t h .

The s p e c i f i t y o f the enzyme a s s a y s was t e s t e d w i t h pure enzymes, s i n g l e and i n m i x t u r e s . M y c e l i a l e x t r a c t s and n u l t u r a l f i l t r a t e s were p r e p a r e d and c o n c e n t r a t e d a t 4°C, e l e c t r o p h o r e s e s and enzyme a s s a y s were c o n d u c t e d a t 25°C.

A l l d e t e r m i n a t i o n s on p e t r i - d i s h e s were done i n t r i p l i c a t e , f o r t h e a s s a y s i n l i q u i d c u l t u r e a l w a y s 10 p a r a l l e l f l a s k s were used.

H e r b a r i u m s p e c i m e n s o f s p e c i e s and s t r a i n s t r e a t e d i n t h i s paper a r e d e p o s i t e d i n the B o t a n i s c h e Staatssammlung Munchen (M) t o g e t h e r w i t h n o t e s on the f r u i t b o d i e s .

A c k n o w l e d g m e n t s : We g r a t e f u l l y acknowledge t h e r e ­c e i p t o f t h e f o l l o w i n g s t r a i n s from: P r o f . J . A r i t a ( T o t t o r i mush­room I n s t . , J a p a n ; No 122/ l u ) , P. C o s t a ( P l e o s , I t a l y ; No 311-2/2v), Dr. K. M o r i ( M o r i mushroom r e s e a r c h I n s t . , J a p a n ; No 4612/ l w ) , J . S c h l i e m a n n (Mykofarm, Hamburg; No P f l 1/ 4b; " F l o r i d a " ) and Dr. M. S e m e r d z i e v a , Prague, CS5R ( 4 r ) .

We a r e a l s o i n d e b t e d t o G. Fuhrmann, R a s t a t t and A. Runge, Munster f o r f r e s h specimens o f P l e u r o t u s .

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B e a d l e G.UI. & E.L. Tatum 1941: G e n e t i c c o n t r o l of b i o c h e m i c a l r e a c ­t i o n s i n N e u r o s p o r a . P r o c . N a t l . A c a d . S c i . USA. 21_'. 499-506.

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H a r k i n , J.IYl., (Yl.J. L a r s e n & J.R. Obst 1974: Use o f s y r i n g a l d a z i n e f o r d e t e c t i o n o f L a c c a s e i n s p o r o p h o r e s o f wood r o t t i n g f u n g i . fYlycologia J56: 469-476.

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Lamoure, D. 1972: A g a r i c a l e s de l a zone a l p i n e . Genre C l i t o c y b e . T r a v . S c i e n t . P a r c N a t i o n a l de l a U a n o i s e 2: 107-152.

Leonowicz A. & 3. T r o j a n o u j s k i 1975a: I n d u c t i o n of a new l a c c a s e form i n t h e fungus P l e u r o t u s o s t r e a t u s by f e r u l i c a c i d . fYlycrobios 13: 167-174.

Leonowicz A. & 3, T r o j a n o w s k i 1975b: I n d u c t i o n o f l a c c a s e by f e r u l i c a c i d i n b a s i d i o m y c e t e s . A c t a b i o c h . P o l o n . 22: 291-295-

L y r , H. 1958: Uber den Nachweis won O x i d a s e n und P e r o x i d a s e n b e i hbheren P i l z e n und d i e Bedeutung d i e s e r Enzyme f u r d i e Bavendamm-Reaktion. P l a n t a . 50: 359-370.

M a c a y a - L i z a n o A . l i . 1974-1975: P l e u r o t u s o s t r e a t u s ( J a c q . e x F r . ) Q u e l e t , formes et especes a f f i n e s . Comportement c u l ­t u r a l et sy s t e m a t i q u e . Rev/, de fYlycol. _39: 3-42.

M o l i t o r i s , H.P. & K. E s s e r 1971: The p h e n o l o x i d a s e s of the ascomycete Podospora a n s e r i n a . V I I . Q u a n t i t a i v e changes i n the spectrum of p h e n o l o x i d a s e s d u r i n g growth i n submerged c u l t u r e . A r c h . l Y l i k r o b i o l . 77: 99-110.

N e a l s o n , K.H. & E.D. Garber 1967: An e l e c t r o p h o r e t i c s u r v e y o f e s t e ­r a s e s , p h o s p h a t a s e s , and l e u c i n e a m i n o p e p t i d a s e s i n m y c e l i a l e x t r a c t s of s p e c i e s of A s p e r g i l l u s . lYlycologia 59_: 330-336.

N o b l e s , IY1.K. 1965: I d e n t i f i c a t i o n o f c u l t u r e s of w o o d - i n h a b i t i n g hy-menomycetes. Can.O.Bot. 4j3: 1097-1139.

O r n s t e i n , L. & B . J . D a v i s 1964: D i s c e l e c t r o p h o r e s i s I u. I I . Ann.N. Y . A c a d . S c i . 121: 321-349, 404-427.

P r i l l i n g e r , H. 1976: G e n e t i s c h e K o n t r o l l e der P h e n o l o x i d a s e " L a c c a s e " des Ascomyceten Podospora a n s e r i n a . B i b l i o t h e c a myco-l o g i a , 51: Cramer, L e h r e .

Prud'Homme, H. 1965: I n : " I n c o m p a t i b i l i t y of F u n g i " 48-52, ed. E s s e r u Raper. C i t a t i o n a f t e r B u r n e t t , O.H. (1968): Funda­m e n t a l s of (Ylycology, A r n o l d p u b l . , London.

P u t t e r , J . 1974a: i n fYlethoden der e n z y m a t i s c h e n A n a l y s e (Bergmeyer, H.U. e d . ) , 725-731, Werlag Chemie, Weinheim.

P u t t e r , 3. 1974b: i n Methods o f E n z y m a t i c A n a l y s i s (Bergmeyer, H.U. ed.) p. 685, V e r l a g Chemie, Uieinheim & Academic Press, New-York, London.

Reddy, IY1.IY1. & S.F.H. T h r e l k e l d 1971: G e n e t i c s t u d i e s of isozymes i n Ne u r o s p o r a I.A. study o f e i g h t s p e c i e s . Can.J.Genet. C y t o l . K3: 298-305.

R'dsch, R. & UU. L i e s e 1970: R i n g s c h a l e n t e s t m it h o l z z e r s t d r e n d e n P i l ­z e n . I . P r u f u n g von S u b s t r a t e n f u r den Nachweis von P h e n o l o x i d a s e n . Arch . ( Y l i k r o b i o l . 73: 281-292.

Romagnesi, H. 1959: Sur l e s P l e u r o t u s du groupe O s t r e a t u s (Ostreomy-ces P i l a t ) . B u l l . S o c . M y c o l . F r a n e e 85: ( 3 ) , 305-314.

S c h i u e i z e r , J . 1919: D i e k l e i n e n A r t e n b e i Bremia L a c t u c a e R e g e l und i h r e A b h a n g i g k e i t won M i l i e u - E i n f l u s s e n . \ / e rh.thur-g a u i s c h . N a t u r f . G e s . 23: 1-61.

S t a n k o v i c o v a , L. 1973: Hyphal s t r u c t u r e i n some p l e u r o t o i d s p e c i e s of A g a r i c a l e s . Nova H e d w i g i a 24_: 61-120.

S w i e z y n s k i , K.M. 1963: Genet. P o l o n i a e _4: 21-36. C i t e d a f t e r B u r n e t t , J.H. ( 1 9 6 8 ) : Fundamentals o f My c o l o g y , A r n o l d p u b l . , London.

S n y d e r , R.D. & C.L. Kramer 1974: P o l y a e r y l a m i d e g e l e l e c t r o p h o r e s i s and n u m e r i c a l taxonomy o f T a p h r i n a c a e r u l e s c e n s and T a p h r i n a deformans. M y c o l o g i a , 66: 743-753.

T y r e l l , D. 1969: B i o c h e m i c a l s y s t e m a t i c s and f u n g i . Bot.Rev. _35: 305-316.

V a n d e n d r i e s , R. 1933: De l a w a l e u r du b a r r a g e s e x u e l , comme c r i t e r i u m dans l ' a n a l y s e d'une sp o r e e t e t r a p l a i r e de b a s i d i o -mycete: P l e u r o t u s o s t r e a t u s . G e n e t i c a K5: 202-212.

Waughan, J.G. 1975: P r o t e i n s and taxonomy i n : Proc.Phytochem.Soc., Symposium, Ghent, B e l g i u m 1973 (Harbone, J.B. and C.F. Wan Sumere, eds.) Academic P r e s s , New Y o r k .

Wang, S.S. & 3.R. Raper 1969: P r o t e i n s p e c i f i c i t y and s e x u a l morpho­g e n e s i s i n S c h i z o p h y H u m commune. J . B a c t . 99: 291-297.

Wang, S.S. & N.A. Anderson 1972: A g e n e t i c a n a l y s i s o f s p o r o c a r p p r o ­d u c t i o n i n P l e u r o t u s s a p i d u s . M y c o l o g i a _64: 521-528.

W r i g l e y , C. 1968: G e l e l e c t r o f o c u s i n g - a t e c h n i q u e f o r a n a l y z i n g m u l t i p l e p r o t e i n samples by i s o e l e c t r i c f o c u s i n g . S c i e n c e T o o l s _15: 17-23.

Z a d r a z i l , F. & M. S c h n e i d e r e i t 1973: D i e Gr u n d l a g e n f u r d i e I n k u l t u r -nahme e i n e r b i s l a n g n i c h t k u l t i v i e r t e n P l e u r o t u s - A r t . Der Champignon 135: 25-32.

1 Pleurotus cornucopiae Paul, ex Fr. ss. Romagn.: strain 4r. 2 Pleurotus "cornucopiae": strain 2v. 3 Pleurotus columbinus Quel, apud Bres.: strain In. A Pleurotus columbinus Quel, apud Bres.: strain In.

8

5 Pleurotus pulmonarius Fr.: strain l r (f rfruit bodies). 6 Pleurotus pulmonarius Fr. ("Florida"): strain 4b. 7 Pleurotus pulmonarius Fr. ("Florida"): strain 4b. 8 Pleurotus ostreatus (Jacqu. ex Fr.) Kummer: strain lw (left), strain Is (right).

9 Pleurotus ostreatus (Jacqu. ex Fr.) Kummer. dark fruit bodies, f,-generation from Is X lw. 10 Pleurotus ostreatus (Jacqu. ex Fr.) Kummer: light fruit bodies, f rgeneration from Is X lw. 11 Pleurotus ostreatus (Jacqu. ex Fr.) Kummer: strain l u ; young fruit body. 12 Pleu­rotus ostreatus (Jacqu. ex Fr.) Kummer: strain l u ; older fruit body.

F i g . 2: £. o s t r e a t u s ( l w ) : Trama o f g i l l s from f r u i t b o d i e s c u l t i v a ­t e d on wheat g r a i n s (a-b) and on wheat s t r a w ( c - d ) . Note s c l e r i f i e d hyphae i n c and d.-b and d m a g n i f i e d s e c t i o n s o f a and c.

K e y

1 ( 2 ) Stipe dark brown in contrast with the cap which i s much li g h t e r . In the Mediterranean: S i c i l y

2 ( 1 ) Stipe not dark brown; equalling in color to the cap or even l i g h t e r

3 ( 6) G i l l s decurrent on long, +_ centric to excentric stipes and cap at the same time +_ infundibiliform

4 ( 5 ) Spores e l l i p t i c a l at the inner side being s l i g h t l y convex. - Cap infundibiliform, with +_ l i g h t or deeper brownish colors. G i l l s running down the stipe reaching the base, being there repeatedly connected by anasto­moses; f i r s t white then changing color to cream and becoming yellowish i f dried. Stipe nearly centric or excentric, often f a i r l y long, 3- 6.5 x 0.8 - 2.3 cm. Spore deposit pale and dingy greyish l i l a c . Odor s p e c i f i c , pleasant sweetish to nearly disagreeable, at the base of the stipe also farinaceous. Hyphae mostly with thin walls, partly also with increasingly thick walls (so c a l l e d false skeletal hyphae). F r u c t i f i c a t i o n partly early in the season

5 ( 4 ) Spores c y l i n d r i c at the inner side being concave or straight. Cap + umbilicate according to the age; li g h t brownish-grey colored; covered by whitish scales, with: the depressed center also with white f e l t . G i l l s running down the long stipe (5 - 6.5 x 0.5 - 1 - 2 cm) to the base, connected by crossveins and forked at least near the top of the stipe; of the same color l i k e the cap, getting orange - ochraceous in older specimens. Mycelium with strong anislike odor.

6 ( 3 ) G i l l s not strongly decurrent and cap not at the same time infundibiliform. Stipes often l a t e r a l l y attached. Spores at the inner side being concave or straight

7 ( 8 ) Cap with l i g h t colors even when young: whitish, pale brownish or pale grey. Trama partly with skeletal hyphae, the walls of which are 1 - 3.5 um thick. G i l l s strongly decurrent, without anastomoses at the top of 1 stipe; f i r s t cream colored, la t e r pale lemon yellowish tinged.

P. cornucopiae Pahl.ex Fr. ss. Romagn.

"P. cornucopiae ?

Stipe often very short, 1 - 1.5 x 0.3 - 0.8 cm, rarely longer. Spore deposit greyish white, sometimes getting l i l a c grey when dried. Odor comparable with that of 4 and 5, sweetish, however not ao strong. F r u c t i f i c a ­tion s t a r t s early i n the season (May, July), partly also l a t e r (e.g. November)

P. pulmonarius Fr. On Opuntia i n the Mediterranean: P. opuntvae

8 ( 7 ) Cap with deeper colors and trama (mostly?) lacking skeletal hyphae. F r u c t i f i c a t i o n late i n the season

9 (10) Cap i f young dark steelblue to l i g h t blue; when older more and more ochraceous starting from the center and leaving the margin of the cap greyish blue, blue greenish or dove-colored. G i l l s whitish with a weak l i l a c tinge, getting yellow patches and f i n a l y somewhat ochraceous; p a r t l y connected by anastomoses near the top of the stipe. Stipe l a t e r a l l y attached, claviform to fusiform, l i g h t e r i n color than the cap, however with comparable colors; strigose at the base, 1 - 2 x 0,5 cm. Spore deposit white.

10 ( 9) Cap not remaining blue at the margin while the center changes color to ochraceous; uniformly colored, most­l y brownish to greybrown

11 (12) On Salix and Populus; separating features against P. ostreatus s t i l l unknown

"P. salignus"(Pers.ex Fr.)

12 (11) On d i f f e r e n t kinds of wood including that of conifers and frondose trees ( i n c l . S a l i x ) . - Cap deep brown, greyish brown, black brown; p a r t i a l l y also rather pale i n older stages of development. G i l l s whitish to s l i g h t l y brownish or grey, decurrent and covering the upper 1/3 of the stipe. Stipe 1.2 - 9 x 0.7 - 2.7 cm, whitish or with s l i g h t brownish tinge, sometimes strigose at the base, mostly d i s t i n c t excentric or even l a t e r a l . Spore deposit whitish to cream colored whith s l i g h t incarnate tinge. Context with strong odor resembling that of some species of Polyporus.

P. columbinus Quel.ap.Bres.

P. ostreatus (Jacqu.ex Fr.) Kummer

Bresadola)

length of spores

i g . 3: Frequency o f spore l e n g t h i n P l e u r o t u s o s t r e a t u s , s t r a i n 2 y ,

on v a r i o u s s u b s t r a t e s . The v a l u e s i n b r a c k e t s i n d i c a t e ave­rage s p o r e l e n g t h .

Merkmalsdivergenz und I n t e r s t e r i l i t a t b e i C l i t o c y b e zusammengestel1t nach E r g e b n i s s e n von D. Lamoure, 1965 und 1972

C. p a u s i a c a H e r k u n f t : Schwedisch Lappland

Inte

r-|

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t

1b C. p a u s i a c a H e r k u n f t : F r a n z o s i s c h e r J u r a

C. c a n d i c a n s +

2b C. c a n d i c a n s v a r . d r y a d i c o l a Sporen: 5 x 3 , 5 /urn Vorkommen: mit v e r s c h . Baumen der W a l d s tufe und im s u b a l p . B e r e i c h , z.T. mit Helianthemum

p - K r e s o l : m i t t l e r e Reakt.

+ Sporen 5-5,5 x 3,5 - 4 Aim Vorkommen: i n der a l p i n e n S t u f e m it Dryas

p - K r e s o l : s t a r k e Reakt.

C. g r a c i l i p e s -

3b C. c a n d i c a n s v a r . d r y a d i c o l a T-amellen weniger gedrangt, b a l d b e i g e f a r b e n . Sporen: 4,5-5,5 x 3,5-4,5 /urn

- L a m e l l e n sehr gedrangt, r e i n weiG, s p a t e r g e l b l i c h Sporen: 4-5 x 3-3,5 yum Stamme aus Lappland und den Alpe n i n t e r f e r t i l

C. f e s t i v a Hut 1,2 - 3,5 cm Frk e i n 7 e l n wachsend S t i e l o b e r f l a c h e n i c h t f a s e r i g , h e l l Geruch: k a m p f e r a r t i g Vorkommen: mit Dryas

Sporen: 5-6 x 3-4 >um

4b C. f e s t i v o i d e s Hut 1,1 - 5,1 cm Frk i n Gruppen und Ringen S t i e l o b e r f l a c h e f a s e r i g , d u n k e l e r g e f a r b t Geruch: sehr unangenehm Vorkommen: mit S a l i x r e t i c u ­l a t a und r e t u s a Sporen: 5,5 x 3-4 Aim

C. gibba Sporen: 5,5-6,5 x 3,5-4,5 Aim -

5b C. c a t i n u s Sporen: 7-8 x 5-6 /urn

a) P.p u l m o n a r i u s (1r) Confrontation of monocaryotic mycelia isolated from a single sporocarp

b) P. columbinus (1n) Confrontation of monocaryotic mycelia isolated from a single sporocarp

3 8 11 12 14

2 + + + + + 18 + + + + + 7 + + + + + 10 + + + •+ +

2 18 7 10|

+ + + + + + + + +

+ + +

+ + +

15 9 1

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4 7 11

3 6 10

+ + + + + + + + +

5 9 12

3 6 10 5 9 12

c) P. p u l m o n a r i u s (1r) x P. co l u m b i n u s ( 1 n ) d) P.p u l m o n a r i u s x P . o s t r e a t u s

A,B. -1"2 A2 B1 V l A2 B2 9 12*8 1 1 7 11*3 6 10*- in

1 2 3 4 5 6 7 8 9 10 l l * - l u I

_. _ o 2 7

J O 3

J3 5 9 1

1 _ • - • _ ! _ . . 2 -' -' -' c 3 o - - - -

t Id (pulmonarius)

3 5 8 11 21 22 23 24 30 « — l r _, , , , , , , , , (pulmonarius)

Is (ostreatus)

+ = clamp connections formed - = no clamp connections formed ' = with barrage phenomenon (see fig.4) o = o u t f a l l of confrontation equivalence of mating factors in interspecies crosses could not be determined

Fig. 4: P.columbinus (In) x P.yulmonarius (lr) Barrage phenomen i n interspecies crosses:

Upper row from l e f t to rig h t : 11 x 13; 6 x 8

Row below from l e f t to rig h t : 12 x 10; 6 x 7

Table 2: Survey of non f e r t i l e crosses of Pleurotus: 1)

cornucopiae X pulmonarius (4r X 4j) 1) cornucopiae X columbinus (4r X In)

X ostreatus (4r x lu)

pulmonarius X 1) cornucopiae

(4j x 4r) pulmonarius X oolumbinus ( l r x 2w) X ostreatus ( l r x lu)

pulmonarius X ostreatus (4b X l a , "Stamm F l o r i d a " oolumbinus X 1)

cornucopiae (In X 4r)

X pulmonarius (In X l r ) X ostreatus (In X lw)

ostreatus X cornucopiae (lu X 4r) X pulmonarius (lu X lr )

(Is X 4x) X columbinus (lw X In)

l u )

1) ss.Romagn. non f e r t i l e = no clamp connections formed

a) P. p u l m o n a r i u s ;

4i 1 o + + o + + - + - o -2 + o + + + + - + + + 3 + + + + + + o - + + + + 4 + + + + + + + - + -

1 2 3 4 5 6 7 8 9 10 11 12. 1 + + + + o + + + + + + + 2 o o + + + + + + + + + o 3 + + + + + + + + + + + + 4 + + + + - + + + -* + + 5 + + + + - + + + + t 4j Origin: Bayerischer Wald; Zwieseler Wald-

haus; 4i und 4j growing 1 km appart from 4h

b) P. p u l m o n a r i u s :

3 8 7 1 18 19 20 24 29 30 1 + + + + + + + + ( + ) + 3 + + + + + + + + + + 4 + + + + + + + + + 5 + + + + + + + + + + 6 + + + + ( + ) + + + + + 7 + + + + + + + + + 8 + + + + + + + ( + ) + + 9 + + + + + + + + + 11 - + + + + + + + + + 12 + + + + + + + + + + t 4h Origin: 4h: Bayerischer Wald, Zwieseler

Waldhaus; l r : Osterreich, Almsee near Scharn-

stein

c) P. p u l m o n a r i u s :

10 11 12 13 14 15 16 17 18 19<-4b 1 + + +'+ + + + + + + 2 + + + + + + + + + + 4 + + + + + + + + + + 6 + + + + + + + + + + 7 + + + + + + + + + + 8 + + +' + + + + + + + 9 + + + + + + + + + + 10 + + +'+ + + + +'+ + 11+ + + 0 + + + +' o + 16 + + + + + + + +' + + l r

Origin: l r : 5sterreich, Almsee near Scharnstein; 4b: North America; "strain F l o r i d a "

d) P. o s t r e a t u s :

lw

1 + + + + + 2 + + + + + 3 +1 + 4 + + + + + 5 +1 + • + 7 + + + ' + + 8 + + + + + 9 + + + + +

1 2 3 4 6 Is 1 + + + + + 2 + + + + + Origin: 3 + + + + + Is: Westfalen

lw: Japan 4 + + + + + 1u: Japan 5 + + + + + 6 + + + + + 7 + + + + + 8 + + + + + 9 + + + + + 10 + + + + +

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DISCUSSION FOLLOWING DRS. BRESINSKY, HILBER AND fYlOLITORIS' PAPER

CLEMENCON: I j u s t wanted t o add a s m a l l o b s e r v a t i o n o f my own. You showed us t h a t t h e s u b s t r a t e , P i c e a or S a l i x , i n f l u e n c e s t h e s p o r e l e n g t h . I was p l a y i n g around w i t h Hypholoma (or Naematoloma) f a s c i c u -l a r e c o l l e c t e d from n a t u r e , and I found c o n s i s t e n t l y t h a t t h e s p o r e s c o l l e c t e d from f r u i t i n g b o d i e s growing on c o n i f e r s were l o n g e r t h a n s p o r e s from f r u i t i n g b o d i e s g r o w i n g on Fagus. I was so much t r o u b l e d t h a t I was a s k i n g m y s e l f , a r e t h e r e two d i f f e r e n t s p e c i e s or n o t , but i n view o f what you a r e t e l l i n g us I t h i n k we have not two d i f f e r e n t s p e c i e s . The d i f f e r e n c e s were r e l a t i v e l y s m a l l but s t a t i s t i c a l l y s i ­g n i f i c a n t .

BAS: I have the i m p r e s s i o n t h a t i n t h e P l e u r o t u s t h a t I a l w a y s used t o c a l l P_. o s t r e a t u s , but I t h i n k now t h a t i t has t o be c a l l e d £. co­l u m b i n u s . a t t h e end o f t h e y e a r , O c t o b e r , november, s p o r e s a r e g e t ­t i n g l o n g e r . I c o l l e c t t h e s e f u n g i a l o n g t h e c o a s t , and I a l w a y s t h o u g h t t h e s p o r e s a r e g e t t i n g l o n g e r because of h i g h e r h u m i d i t y , but t h i s i s j u s t an i m p r e s s i o n . Another t h i n g I n o t i c e d : when you keep them t o o l o n g i n t h e r e f r i g e r a t o r , t h e s p o r e s a r e g e t t i n g l o n g e r a l ­so .

UATLING: I have measured l i t e r a l l y t h ousands of s p o r e s i n v a r i o u s members o f t h e B o l b i t i a c e a e t o see t h e s o r t of e f f e c t one g e t s w i t h d i f f e r e n t s u b s t r a t e s e t c . ; I t h i n k i n the B o l b i t i a c e a e changes may be extreme, because th e f r u i t b o d i e s have very t h i n f l e s h and whatever happens t o t h e e nvironment i s i m m e d i a t e l y e x p r e s s e d i n t h e s p o r e s . I n f a c t , i n B o l b i t i a c e a e we a l s o see a s l i g h t change i n shape. I f you measure the Q-Value f o r t h e s p o r e d e p o s i t you f i n d t h e r e t o o you get not o n l y a s h o r t e n i n g of t h e s p o r e s or a l e n g t h e n i n g of the s p o r e s , but a l s o a change i n shape; t h i s change i n shape may a l s o be c o r r e l a ­t e d w i t h age o f the f r u i t i n g body. These a r e not i s o l a t e d c a p s , t h i s i s a f r u i t i n g body which i s s t i l l g r o w i n g .

CLEIY1ENC0N: I d i d t h i s w i t h many f u n g i , a l s o w i t h f l e s h y f u n g i , l i k e Hebeloma l e u c o s a r x t h a t i s g r o w i n g i n my b a c k y a r d , and I n o t i c e d one v e r y i n t e r e s t i n g t h i n g . U s u a l l y t h e l e n g t h of t h e s pore and t h e width, t h a t means a l s o the volume, are i n c r e a s i n g ; t h e n d e c r e a s i n g . Once I had a s t r o n g n o r t h wind t h a t somewhat d r i e d the mushrooms. And even

i n the f l e s h y f r u i t b o d i e s t h e s p o r e volume, the l e n g t h and w i d t h , dropped s h a r p l y and came back a g a i n when normal c o n d i t i o n s were r e s ­t o r e d .

UJATLING: Y e s , you can a l t e r the s p o r e s w i t h t h e e n v i r o n m e n t a l c o n d i ­t i o n s .

CLEMENCON: And even i n f l e s h y f u n g i , not o n l y i n t h i n t h i n g s .

UJATLING: That i s v e r y i n t e r e s t i n g , because I a l w a y s thought t h a t i n s p e c i e s l i k e T r i c h o l o m a o r o t h e r f l e s h y f r u i t i n g b o d i e s t h i s would not be so much t h e c a s e .

BRESINSKY: To what e x t e n d do you have t h o s e d i f f e r e n c e s ? How many mi c r o n s ?

UJATLING: Oh! 3 m i c r o n s !

SMITH: What about t h e c o l o r o f the s p o r e d e p o s i t ?

BRESINSKY: I d i d not i n c l u d e t h i s c h a r a c t e r h e r e . Of c o u r s e we n o t e d t h e c o l o r o f s p o r e p r i n t , but we d i d not i n c l u d e i t i n any e x p e r i m e n ­t a l s e t u p so f a r . C e r t a i n l y i t w i l l be a v e r y i m p o r t a n t f e a t u r e , and I know t h a t you i n t h e S t a t e s have d i f f e r e n t s p e c i e s , P_. s a p i d u s and JP. o s t r e a t u s , t h a t a r e p r e t t y w e l l d i s t i n g u i s h e d by d i f f e r e n t c o l o r s o f t h e s p o r e p r i n t . T h i s does a p p a r e n t l y not work i n Europe.

SMITH: Does your dark P l e u r o t u s o s t r e a t u s have a d i s t i n c t l y c o l o r e d spore d e p o s i t ?

BRESINSKY: M o s t l y i t s t a r t s w i t h a c o l o r w h i t i s h and t h e n we get more and more l i l a c c o l o r s .

SMITH: Y e s , you have not worked on t h i s w h i t i s h o s t r e a t u s - l i k e f u n ­gus t h a t we have from our B i o l o g i c a l S t a t i o n i n N o r t h e r n M i c h i g a n ?

BRESINSKY: No, I have n o t . I would l i k e t o have s p o r e s .

BLAICH: You t o l d us about s e g r e g a t i o n o f t h e cap c o l o r , UJhat t y p e of s e g r e g a t i o n ? One t o one?

BRESINSKY: So f a r as me c o u l d make c a l c u l a t i o n s , on a l i m i t e d mate­r i a l o n l y , i t i s one t o one.

BLAICH: So here i s c e r t a i n l y t h e p o s s i b i l i t y o f a monogenic d i f f e ­r e n ce between t h e two s t r a i n s . And t h i s would be a d i f f e r e n c e which i s c l e a r l y not a s p e c i f i c d i f f e r e n c e .

ESSER: I was v e r y p l e a s e d t o see t h a t you have o b s e r v e d about the same phenomena i n your t U e s t f a l e n s t r a i n , where does i t come from?

BRESINSKY: lYlunster, U J e s t f a l e n , leg.A.Runge 13.12.1973; i s o l a t e d by 0. H i l b e r 17.12.1973.

ESSER: You use t h e word " i n t e r s t e r i l i t y " . And I have a tendency t o l o o k c a r e f u l l y a t i n t e r s t e r i l i t y , because i n t e r s t e r i l i t y might be t r u e s t e r i l i t y , but i t might be due t o o t h e r mechanisms which have n o t h i n g t o do w i t h s t e r i l i t y , l i k e h e t e r o g e n i c i n c o m p a t i b i l i t y . At the moment I would say f a i l u r e o f i n t e r f e r t i l i t y , I would not say i n ­t e r s t e r i l i t y .

WATLING: I do not know whether our l a s t s p e a k e r s u f f e r e d from t h e r e s t l e s s n i g h t s t r y i n g t o put t o g e t h e r h i s e x p e r i m e n t a l work and h i s f i e l d work as I have because i n t h e B o l b i t i a c e a e we have s i m i l a r v a ­r i a b i l i t i e s o f s p o r e s i z e on d i f f e r e n t s u b s t r a t e s . When you go i n t o t h e f i e l d you do not f i n d t h e same degree o f v a r i a b i l i t y . I n t h e f i e l d I r e a l l y n ever r e a c h e d t h e extremes so t h a t i n f a c t n a t u r e seems t o put a b u f f e r i n g e f f e c t on v a r i a t i o n . You d i d mention s e l e c ­t i o n . There i s a s t r a i n o f P s i l o c y b e m e r d a r i a which was found i n 1968; a t t h e base o f t h e f r u i t i n g body s m a l l g a s t r o i d f r u i t i n g b o d i e s were o b s e r v e d . They d i d not have a l l t h e normal c h a r a c t e r i s t i c s , and by s e l e c t i o n of t h e s e f r u i t i n g b o d i e s , and not t h e a g a r i c o i d f r u i t i n g b o d i e s , a f t e r t h r e e g e n e r a t i o n s a c u l t u r e was produced which was ba­s i c a l l y g a s t r o i d .

BRESINSKY: A p u b l i s h e d paper?

UJATLIIMG: Yes, New P h y t o l o g i s t 70: 307-326, 1971. The problem, o f c o u r s e , i s whether i t i s c y t o p l a s m i c a l l y i n h e r i t e d , and a t t h e moment we a r e i n v e s t i g a t i n g t h i s p o s s i b i l i t y ; i s i t a v i r u s p a r t i c l e which

i n f a c t iue a r e s e l c t i n g f o r each t i m e we s e l e c t t h e s e " a b e r r a n t " f r u i t i n g b o d i e s ? I t so we f i n i s h up w i t h a c u l t u r e which g e t s more and more of t h i s v i r a l "compound"; i t i s a h y p o t h e s i s , but we have ye t t o f i n d the v i r u s p a r t i c l e s .

SINGER: I u n d e r s t a n d t h a t M c K n i g h t , who f i r s t produced such g a s t r o i d f r u i t i n g b o d i e s i n P s i l o c y b e , c o n s i d e r s t h i s now as a m u t a t i o n t r i g ­g ered by t h e c o n d i t i o n s .

UJATLING: From comparing M c K n i g h t ' s e x p e r i m e n t a l work i t c e r t a i n l y J o es not l o o k t o be t h e same t h i n g . My m a t e r i a l has been passed over to a g e n e t i c i s t who i s much more competent t h a n I t o work out t h e p r o b l e m . P r o f e s s o r B u r n e t t now has c u l t u r e s of a l l the d i f f e r e n t s t r a i n s w h i ch we have been a b l e t o s e l e c t o u t . H o p e f u l l y i n t h e f u ­t u r e we w i l l have some e v i d e n c e i n t h e B a s i d i o m y c e t e s o f s e l e c t i o n o f t h i s t y p e .

ROMAGNESI: Mo n s i e u r B r e s i n s k y , vous avez pu f a i r e ce que j e n ' a i pas pu f a i r e a mon grand r e g r e t . Je v o u d r a i s s u r t o u t vous demander q u e l -ques p r e c i s i o n s en me p l a c a n t s u r t o u t du p o i n t de vue du s y s t e m a t i -c i e n . Je d o i s vous donner mon a c c o r d complet en ce q u i c o n c e r n e l a p r e s e n c e d'hyphes a p a r o i e p a i s s i e dans l a trame du P l e u r o t u s pulmo­n a r i u s . J ' a i t o u j o u r s et& pr^occupe" de s a v o i r s ' i l s ' a g i s s a i t d'un c a r a c t e r e c o n s t a n t . P a r c e que j e r e c o l t a i s souvent dans l a n a t u r e des c a r p o p h o r e s , ou des b a s i d i o m e s , s i vous p r e f e r e z , q u i a v a i e n t t o u s l e s c a r a c t e r e s e x t ^ r i e u r s de p u l m o n a r i u s sans en a v o i r l e s hyphes e p a i s s i e s . l/ous savez p e u t - e t r e , qu'au L a b o r a t o i r e de C r y p t o g a m i e du Museum de P a r i s , M o n s i e u r Roger C a i l l e u x c u l t i v e a r t i f i c i e l l e m e n t t o u s l e s P l e u r o t u s du groupe o s t r e a t u s , a i n s i que ceux du groupe e r y n g i i . Je l u i a i done c o n f i e une s p o r e e d'un p u l m o n a r i u s & hyphes e p a i s s i e s q u ' i l a cultiv£e sur compost. E t , de f a i t , l e s hyphes a p a r o i s e p a i s s i e s q u i se t r o u v a i e n t dans l e c a r p o p h o r e trouve" dans l a n a t u r e ne se t r o u v a i e n t p l u s , ou t o u t au moins ce c a r a c t e r e 6 t a i t ex-tremement a t t e n u e . I I e s t done c l a i r , comme vous l e p e n s e z , que ce c a r a c t e r e e s t en p a r t i e l i e a 1'environnement. Cependant, j ' a i f a i t p e r s o n n e l l e m e n t dans une f o r e t des e n v i r o n s de P a r i s l a r ^ c o l t e s i -m u l t a n e e , s u r un meme t r o n c de Faqus a b a t t u , l a r ^ c o l t e d'une grande s e r i e de c a r p o p h o r e s ayant des hyphes a p a r o i mince e t , au b o u t , un p e t i t groupe de t r o i s ou q u a t r e c a r p o p h o r e s q u i a v a i e n t des hyphes & p a r o i e p a i s s i e . Dans ce cas i l n ' e s t pas p o s s i b l e d'6voquer l ' e n v i -

ronnement ou des c o n d i t i o n s e x t ^ r i e u r e s p u i s q u ' e l l e s 6 t a i e n t l e s me­rcies. Deuxiemement j e v o u d r a i s vous demander q u e l q u e s p r e c i s i o n s s ur l a fagon dont uous conceuez l e P l e u r o t u s c o l u m b i n u s . E s t - c e que vous avez r e c o l t e c e t t e espece en h i v e r ?

BRESINSKY: £1. colu m b i n u s s h o u l d be a s p e c i e s i n a c c o r d a n c e w i t h B r e s a d o l a , I c o n o g r a p h i a , t a b . 291. Our P I . c o l u m b i n u s has been c o l ­l e c t e d i n November ( i n ; K a r l s r u h e , leg.Fuhrmann) and i n October ( 4 t ; P o l a n d ; l e g . IY1. (Yloser). I don't t h i n k t h a t s c l e r i f i e d hyphae a r e an ab­s o l u t e l y r e l i a b l e c h a r a c t e r f o r P I . p u l m o n a r i u s .

ROfYlAGNESI: De veux vous donner l a r a i s o n de l a q u e s t i o n . Oe ne s a i s s i wous c o n n a i s s e z une t h e s e s u r l e P l e u r o t u s o s t r e a t u s q u i a e t e soutenue, i l y a un ou deux ans, au L a b o r a t o i r e de Cryptogamie de Mon s i e u r Heim a P a r i s . I I s ' a g i s s a i t d'une £tudiante S u d - a m e r i c a i n e , dont j ' a i malheureusement o u b l i e l e nom c o m p l i q u e , mais q u i a w a i t e t u d i e s u r t o u t l e comportement du groupe o s t r e a t u s e t e r y n q i i en c u l ­t u r e p o r t a n t p r i n c i p a l e m e n t sur l ' i n c i d e n c e des c a r a c t e r e s de tempe­r a t u r e e t d ' a u t r e s f a c t e u r s p h y s i o l o g i q u e s q u i accompagnaient ces c u l t u r e s . I I a 6te a i n s i t r o u v e d'une fagon extremement c l a i i e que dans l e s r e c o l t e s de P l e u r o t u s o s t r e a t u s i l y a v a i t deux groupes d'especes q u i se d i f f e r e n c i a i e n t t r e s nettement par l a t e m p e r a t u r e o p t i m a l e du developpement. Et ce que l e s mycologues f r a n g a i s ont t o u -j o u r s c o n s i d e r ^ comme P l e u r o t u s o s t r e a t u s e s t une espece d ' h i v e r dont l'optimum de developpement e s t r e l a t i v e m e n t bas, t a n d i s que pulmona­r i u s , c o r n u c o p i a e et l e s a u t r e s e s p e c e s q u i poussent au p r i n t e m p s ou en automne ont un optimum de t e m p e r a t u r e beaucoup p l u s e i e v e . Or, des formes d ' h i v e r que nous avons t o u j o u r s c o n s i d e r e e s comme e t a n t l e t y ­pe P l e u r o t u s o s t r e a t u s s o n t des formes q u i pr£sentent des c o l o r a t i o n s b l e u e s p u i s q u e l e s c a r p o p h o r e s v i r e n t au brun dans l a v i e i l l e s s e . Oe me demande s i vos P l e u r o t u s c o l u m b i n u s ne s e r a i e n t pas nos P l e u r o t u s o s t r e a t u s au sens des mycologues f r a n g a i s .

BRESINSKY: I am s o r r y t h a t I cannot g i v e a f i n a l answer, s i n c e t h i s q u e s t i o n s h o u l d be s o l v e d on the base of c o n f r o n t a t i o n e x p e r i m e n t s . What we c e r t a i n l y know i s t h a t t h e r e a r e a t l e a s t two d i f f e r e n t d a r k -c o l o r e d s p e c i e s which a r e f r u i t i n g i n t h e l a t e season and which we are c a l l i n g P_. o s t r e a t u s and P. c o l u m b i n u s .

ROfYlAGNESI: Sur 1' i n t e r p r e t a t i o n de P l e u r o t u s c o l u m b i n u s i l y a des d i f f i c u l t y . En e f f e t , j e c r o i s vous r a p p e l e z que l a p l a n c h e d'Icones

de B r e s a d o l a , dans l e s f u n g i t r i d e n t i n i vous avez P l e u r o t u s o s t r e a ­t u s , r e p r e s a n t e un champignon q u i n ' e s t pas b l e u , n i b l e u d ' a c i e r , mais q u i e s t v e r t f u l i g i n e u x .

BRESINSKY: Ce n ' e s t pas o s t r e a t u s , c ' e s t c o l u m b i n u s .

ROfYlAGNESI : Or, i l y a p e u t - e t r e q u a r a n t e annees qu'au j a r d i n des p l a n t e s de P a r i s j ' a i r e c o l t e s u r un C e r c i s s i l i q u a s t r u m ce que j e pense e t r e l e c o l u m b i n u s de B r e s a d o l a . Eh b i e n , ce champignon, l o r s -q u ' i l e s t jeune a un chapeau f a u v e ou o c r e , e t quand i l se d^veloppe i l prend exactement l a c o u l e u r g r i s - o l i v a t r e v e r d a t r e de l a P l a n c h e de B r e s a d o l a . Et s u r l a p l a n c h e meme de B r e s a d o l a l a c o u l e u r r o u g a t r e a l a q u e l l e vous f a i t e s a l l u s i o n e s t t r e s p r o c h e , en e f f e t , de c e l l e que j ' o b s e r v e . D ' a u t r e p a r t , vous savez q u ' i l y a dans l a l i t t e r a t u r e des donn^es sur l a t o x i c i t e de P l e u r o t u s c o l u m b i n u s . Or, j e p u i s vous a f f i r m e r , que l e P l e u r o t u s b l e u q u i pousse en h i v e r e s t p a r f a i t e m e n t c o m e s t i b l e . Je me demande done, s i en f a i t , v o t r e c o l u m b i n u s n ' e s t pas l e v e r i t a b l e o s t r e a t u s et s i v o t r e o s t r e a t u s n ' e s t pas l e cham­pig n o n qu'a t o r t ou a r a i s o n j ' a i donne dans mon t r a v a i l s u r ce g r o u ­pe sous l e nom de s a l i g n u s , l e q u e l e s t en e f f e t un champignon b r u n , et q u i pousse a u s s i b i e n s u r f e u i l l u que s u r c o n i f e r e s . I I a u r a i t l a p e u t - e t r e un probleme de s y s t e m a t i q u e a r e s o u d r e .

BRESINSKY: Some of t h e s p e c i e s , l i k e P l e u r o t u s c o l u m b i n u s , a r e v e r y ambiguas. T h i s shows t h a t b r e e d i n g e x p e r i m e n t s a r e n e c e s s a r y here i n o r d e r t o f i n d t h e b o r d e r l i n e s between t h e s p e c i e s and i n o r d e r t o d e c i d e what i s c o n s p e c i f i c or n o t . UJe s h o u l d have t h e o p p o r t u n i t y t o i n v e s t i g a t e your PJ^. s a l i g n u s .

BLAICH: Are t h e r e b r e e d i n g e x p e r i m e n t s columbinus - 0 3 t r e a t us?

BRESINSKY: Columbinus x o s t r e a t u s a r e i n p r e p a r a t i o n .

SfYlITH: I am w o n d ering i f your s i t u a t i o n here i n Europe i s not c o n f u ­sed somewhat by i n v a d i n g American v a r i a n t s . Now we have c o l u m b i n u s i n t h e w e s t e r n U n i t e d S t a t e s and i t i s b l u e and i t i s on c o n i f e r s . I ha­ve never seen t h i s b l u e t h i n g on a n y t h i n g but c o n i f e r . I t a k e i t you get i t on hardwoods h e r e . In the s p r i n g we have what we c a l l o c h r a c e ­ous t o w h i t e P l e u r o t u s and I t r i e d t o i d e n t i f y i t w i t h a number o f European s p e c i e s . I t has a s p o r e p r i n t t h a t i s w h i t e y e l l o w i s h t o

sometimes p a l e l i l a c . T h i s grows on aspen. T h i s i s t h e one uue a r e g o i n g t o send you. And t h e n we have t h e b i g dark o s t r e a t u s w i t h a r a t h e r b i t t e r t a s t e , i t i s a l m o s t b l a c k and i t grows i n the s p r i n g a f t e r t h e snow m e l t s on t h e s k i s l o p e s . I t i s v e r y d i s t i n c t from t h e o r d i n a r y P l e u r o t u s t h a t we get on t h e elm and o t h e r hardwood.

BRESINSKY: I t h i n k t h a t i t i s v e r y i n t e r e s t i n g t h a t we can o b s e r v e so much v a r i a t i o n i n P l e u r o t u s w i t h i n one s p e c i e s as w e l l as between the s p e c i e s .

ESSER: I t h i n k i n f u t u r e g e n e r a l r e g u l a t i o n s have t o become e f f i ­c i e n t . Some y e a r s ago c o m m e r c i a l f i r m s have s o l d c u l t u r e s o f P l e u r o ­t u s o s t r e a t u s t o everybody who wanted t o b reed t h i s fungus i n h i s basement. A f t e r h a r v e s t i n g the f r u i t b o d i e s t h e p e o p l e has thrown t h e c u l t u r e s s u b s t r a t e on t h e t r a s h p i l e . These r a c e s o f P l e u r o t u s o s t r e ­a t u s became a chance t o i n t e r b r e e d w i t h each o t h e r and w i t h many o t h e r f u n g i . There i s a n o t h e r q u e s t i o n : I have h e a r d t h a t P l e u r o t u s o s t r e a t u s may cause a l l e r g i e s . I s t h i s a rumor? Or i s t h i s p o s s i b l e due t o the f a c t t h a t t h e s p o r e s o f P l e u r o t u s o s t r e a t u s a r e a b l e t o g e r m i n a t e a t tem­p e r a t u r e s up t o 40°C and t h e r e f o r e may cause a f t e r i n h a l i n g i n f l a m m a ­t i o n s o f t h e l u n g ? Are t h e s e a l l e r g i c r e a c t i o n s caused by a l l o s t r e a ­t u s s t r a i n s o r o n l y by c e r t a i n r a c e s ? Or even by o t h e r s p e c i e s l i k e .columbinus?

BRESINSKY: Hie have put i t on our program.

ESSER: What i s known?

BRESINSKY: I t i s known t h a t P l e u r o t u s causes a l l e r g i e s . We don't know wether t h i s i s t r u e f o r a l l s p e c i e s .

BAS: T h i s i s p o s s i b l y a m a t t e r o f t h e amount o f s p o r e s . P l e u r o t u s o s t r e a t u s s t a r t s t o s p o r u l a t e v e r y e a r l y . They have been g r o w i n g P l e u r o t u s o s t r e a t u s i n mushroom houses i n the N e t h e r l a n d s . The wor­k e r s r e f u s e d t o go i n because t h e r e was a m i s t o f s p o r e s . They had t o wear a k i n d o f mask because they c o u l d not s t a n d t h e s p o r e s . I t h i n k i f you i n h a l e c l o u d s o f s p o r e s , t h e r e must be some e f f e c t .

SINGER: The o n l y i n f o r m a t i o n I have i s t h a t i n Ohio t h e r e was an

a l l e r g i c r e a c t i o n .